Purified forms of rofecoxib, methods of manufacture and use

ABSTRACT

The subject matter disclosed herein relates to rofecoxib, also known as TRM-201 or RXB-201, its method of manufacture, and use. In certain aspects, the highly pure or substantially pure rofecoxib as provided herein has a favorable purity profile and is the active ingredient in a pharmaceutical composition that is administered to treat or prevent a number of conditions, including pain associated with a condition caused by a bleeding disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/186,697, filed Feb. 26, 2021 titled PURIFIED FORMS OF ROFECOXIB.METHODS OF MANUFACTURE AND USE, which is a continuation of U.S.application Ser. No. 16/716,242, filed Dec. 16, 2019, titled PURIFIEDFORMS OF ROFECOXIB, METHODS OF MANUFACTURE AND USE, which is acontinuation of International PCT Application Number PCT/US19/61178,filed Nov. 13, 2019, titled PURIFIED FORMS OF ROFECOXIB, METHODS OFMANUFACTURE AND USE, which claims the benefit of and priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 62/770,563, filedNov. 21, 2018, titled HIGHLY PURE ROFECOXIB. METHODS OF MANUFACTURE ANDUSE, the contents of which are incorporated herewith in their entirety.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Rofecoxib is a selective COX-2 inhibitor, nonsteroidal anti-inflammatorydrug (NSAID). that was marketed under the brand name “VIOXX” until itwas withdrawn from the market in 2004 over safety concerns. Before beingwithdrawn from the market, “VIOXX” was approved in the United States forthe following indications: signs and symptoms of osteoarthritis (OA);signs and symptoms of rheumatoid arthritis (RA) in adults; signs andsymptoms of pauciarticular or polyarticular course Juvenile RheumatoidArthritis; management of acute pain in adults; treatment of primarydysmenorrhea; and treatment of migraine attacks with or without aura inadults.

There is a long felt but unmet need in the art for new medications forthe treatment of pain, fever, and inflammation. This is especially truewith respect to subjects suffering from pain that is co-morbid orassociated with diseases or conditions arising from bleeding disorders,including hemophilic arthropathy and von Willebrand's disease.Hemophilia is a bleeding disorder caused by inherited or spontaneousmutations in genes that code for clotting factor. The most common formsof hemophilia are the result of deficiencies in the coagulation factorsVIII (hemophilia A) or IX (hemophilia B). The prevalence of subjectswith hemophilia (PWH) in the United States is estimated at 20,000subjects.

Blood-induced joint disease may follow acute joint injury and isassociated with hemophilia, with intra-articular bleeding (hemarthrosis)accounting for more than 90% of all serious bleeding events in subjectswith severe hemophilia (baseline factor (F)VII or FIX activity <1%).Over time, recurrent bleeding into the same articular joints results inprogressive damage and the development of hemophilic arthropathy.Despite advances in treatment and the delivery of comprehensive care atdedicated centers, joint bleeding and arthropathy remain the singlelargest cause of morbidity in PWH.

Although the pathogenesis of the blood-induced joint injury ofhemophilic arthropathy has not been fully elucidated, it appears to havesimilarities with the degenerative joint damage that occurs inmechanically-induced joint injury such as OA and the inflammatoryprocesses associated with RA.

Arthritis as a result of hemophilia is the most common comorbidity inadult PWH, with 44-55% of subjects reporting suffering from hemophilicarthropathy. Pain/discomfort is the most frequently reported limitationfor PWH, with 75% of PWH reporting this limitation. Eighty-nine percentof PWH reported that pain had interfered with their daily life in thepast 4 weeks, and 50% reported suffering from constant pain.

In additional to its impact on quality of life, hemophilic arthropathyhas a significant impact on the cost associated with the treatment ofhemophilia. As acute pain associated with hemarthrosis and chronic painassociated with arthritis are largely indistinguishable for PWH,significant over-utilization of factor replacement to address painsymptoms have been reported and 58% of PWH report using factorreplacement to treat chronic pain. In addition to being medicallyineffective, this misutilization has significant economic implications,as factor replacement is one of the costliest known drug interventions.

Currently, management of the pain and inflammation associated withhemophilic arthropathy is difficult, due to a lack of approvedtreatments and limitations to existing off-label options. Acetaminophenis generally recommended as first-line therapy, however, it is oflimited utility owing to its lack of anti-inflammatory effect andincreased risk of hepatic adverse events in a subject population with asignificant rate of comorbid chronic hepatitis C.

Opioids can be used to alleviate pain associated with hemophilicarthropathy, but also have little anti-inflammatory effect and long-termuse of this mode of analgesic therapy may lead to tachyphylaxis,dependence, and the potential for abuse. Additionally, a 2010observational study by Solomon et al. demonstrated that long-termchronic use of opioids to treat arthritis in the general populationresulted in increased risk of (cardiovascular) CV events, fractures,hospitalization, and all-cause mortality relative to non-selectiveNSAIDs or a combined group of therapeutic and supra-therapeutic doses ofCOX-2 selective NSAIDs.

Due to their effect on platelet function, NSAIDs, particularlyacetylsalicylic acid (ASA), are not recommended in PWH. In addition,even otherwise trivial NSAID-induced ulcerations might be expected toresult in more severe or prolonged bleeding in the setting of anunderlying coagulopathy, and it has been demonstrated that NSAIDs areassociated with an increased risk of upper gastrointestinal (UGI)complications, including ulcers, bleeding and perforations, in PWH.Thus, there is a long felt but unmet need in the art for medications totreat pain in PWH.

Von Willebrand Disease (vWD) is a genetic disorder caused by defectivevon Willebrand factor, a clotting protein vWD is the most common form ofbleeding disorder, affecting ˜1% of US population, though mild cases areoften undiagnosed. Generally mild and characterized by heavy mucosalbleeding, more severe forms can manifest into significant broad-ranginginternal bleeding. Evidence suggests that vWD subjects are moresusceptible to migraine headaches. Due to risk of exacerbatinghemorrhaging, NSAIDs are not recommended for pain management in subjectswith vWD.

Juvenile idiopathic arthritis, including systemic juvenile idiopathicarthritis (SJIA) is one of several rheumatic diseases that affectchildren. SJIA affects the entire body, including the joints. SJIAtypically occurs in flares with some subjects having healthy periodsbetween flares. Diagnosis may be delayed by these ups and downs over thecourse of the disease. SJIA is generally easier to diagnose during aflare. The most common symptoms of SJIA are recurrent bouts of feverwith high daily temperature spikes, skin rash, and painful, stiffjoints.

SUMMARY OF THE INVENTION

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising highly pure rofecoxib or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In some embodiments, the highly pure rofecoxib comprises less than about0.10%, about 0.075%, about 0.05%, about 0.025%, about 0.02%, about0.01%, or about 0.001% total impurities. In some embodiments, the highlypure rofecoxib is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the highly pure rofecoxib comprises less than about 0.10%,about 0.05%, about 0.02%, or about 0.01% of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. In some embodiments,the highly pure rofecoxib comprises less than about 0.10%, about 0.05%,about 0.02%, or about 0.01% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising substantially pure rofecoxib or anacceptable salt thereof and a pharmaceutically acceptable carrier.

In some embodiments, the substantially pure rofecoxib comprises lessthan about 0.40%, about 0.30%, about 0.25%, about 0.20%, or about 0.15%total impurities. In some embodiments, the substantially pure rofecoxibis substantially free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the substantially pure rofecoxib comprises less than about0.25%, about 0.20%, or about 0.15% of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. In some embodiments,the substantially pure rofecoxib comprises less than about 0.25%, about0.20%, or about 0.15% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

In some embodiments, the highly pure rofecoxib comprises less than about0.10%, about 0.05%, about 0.02%, about 0.01%, or is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one. In someembodiments, the highly pure rofecoxib comprises less than about 0.10%,about 0.05%, about 0.02%, about 0.01%, or is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising an effective amount of highly purerofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the pain, fever, or inflammation is caused by oneor more conditions selected from the group comprising of hemophilicarthropathy, osteoarthritis, rheumatoid arthritis, pauciarticular orpolyarticular course Juvenile Rheumatoid Arthritis (JRA), juvenileidiopathic arthritis, acute pain, primary dysmenorrhea, migraineattacks, or migraine associated with von Willebrand disease. In otherembodiments, the pain or inflammation is caused by psoriatic arthritisor fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising an effective amount ofsubstantially pure rofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the pain, fever, or inflammation is caused by oneor more conditions selected from the group comprising of hemophilicarthropathy, osteoarthritis, rheumatoid arthritis, pauciarticular orpolyarticular course Juvenile Rheumatoid Arthritis (JRA), juvenileidiopathic arthritis, acute pain, primary dysmenorrhea, migraineattacks, or migraine associated with von Willebrand disease. In otherembodiments, the pain or inflammation is caused by psoriatic arthritisor fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain or migraine associated with a condition causedby a bleeding disorder in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of highly pure rofecoxib and apharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain or migraine associated with a condition causedby a bleeding disorder in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of substantially pure rofecoxib and apharmaceutically acceptable carrier.

In some embodiments, the subject is age 2 or older. In some embodiments,the subject is 12 years old to 75 years old. In some embodiments, thesubject does not have a history or current symptoms of cardiovasculardisease. In some embodiments, the subject does not have a history orcurrent symptoms of gastrointestinal bleeding, ulceration, orperforation. In some embodiments, the pharmaceutical composition isadministered once daily. In some embodiments, the pharmaceuticalcomposition is administered two times or more daily. In someembodiments, the condition is caused by a bleeding disorder. In someembodiments, the bleeding disorder is hemophilia A or B, C vonWillebrand Disease, or a drug-induced bleeding disorder. In someembodiments, the condition is hemophilic arthropathy, juvenileidiopathic arthritis, or migraine associated with von Willebranddisease.

In some embodiments, the treatment is effective at treating pain withoutco-administration of an analgesic. In some embodiments, the treatmentresults in a subject decreasing or discontinuing use of analgesics orrescue medications during the treatment as compared to before initiationof the treatment. In some embodiments, the treatment results in asubject decreasing or discontinuing use of opioid medications during thetreatment.

In some embodiments, the method further includes administering agastro-protective agent. In some embodiments, the gastro-protectiveagent is co-administered with the pharmaceutical composition. In someembodiments, the treatment does not include administeringgastro-protective agent.

In some embodiments, the treatment achieves a reduction of at least 1from baseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 2 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 3 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 4 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 5 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the reduction in the Pain Intensity Numerical Rating Scaleis achieved within 1, 2, 3, 4, 5, 6 days, 1 week, or 2 weeks ofadministering the pharmaceutical composition.

In some embodiments, the effective amount of the rofecoxib administeredto a subject is 12.5 mg. In some embodiments, the effective amount ofthe rofecoxib administered to a subject is 17.5 mg. In some embodiments,the effective amount of the rofecoxib administered to a subject is 20mg. In some embodiments, the effective amount of the rofecoxibadministered to a subject is 25 mg.

In some embodiments, the effective amount of the rofecoxib administeredto a subject is selected from the group comprising of 1 mg, 2 mg, 3 mg,5 mg, 6.25 mg, 7.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 ng, 12.5 mg,13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 ng, 15.5 mg, 16 mg, 16.5 mg, 17 mg,17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg,22.5 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg,and 70 mg. In some embodiments, the effective amount of the rofecoxibadministered to a subject is selected from the group comprising of 0.10mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, or0.70 mg/kg.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering once daily to the subject a pharmaceutical compositioncomprising 12.5 mg of substantially pure rofecoxib or 12.5 mg of highlypure rofecoxib as a sole active ingredient, wherein the treatmentachieves a reduction of at least 1 from baseline in a Pain IntensityNumerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering once daily to the subject a pharmaceutical compositioncomprising 17.5 mg of substantially pure rofecoxib or 17.5 mg of highlypure rofecoxib as a sole active ingredient, wherein the treatmentachieves a reduction of at least 1 from baseline in a Pain IntensityNumerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering once daily to the subject a pharmaceutical compositioncomprising 20 mg of substantially pure rofecoxib or 20 mg of highly purerofecoxib as a sole active ingredient, wherein the treatment achieves areduction of at least 1 from baseline in a Pain Intensity NumericalRating Scale.

In some embodiments, the pharmaceutical composition achieves a reductionin a Pain Intensity Numerical Rating Scale that is equal to or greaterthan the once daily administration of a pharmaceutical compositioncomprising 25 mg of rofecoxib that is not highly pure. In someembodiments, the condition is hemophilic arthropathy.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with hemophilic arthropathy in asubject in need thereof, wherein the subject is 12 years old or older,the method comprising administering once daily a pharmaceuticalcomposition comprising 12.5 mg of substantially pure rofecoxib or 12.5mg of highly pure rofecoxib, wherein the treatment achieves a reductionof at least 1 from baseline in a Pain Intensity Numerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with hemophilic arthropathy in asubject in need thereof, wherein the subject is 12 years old or older,the method comprising administering once daily a pharmaceuticalcomposition comprising 17.5 mg of substantially pure rofecoxib or 17.5mg of highly pure rofecoxib, wherein the treatment achieves a reductionof at least 1 from baseline in a Pain Intensity Numerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with hemophilic arthropathy in asubject in need thereof, wherein the subject is 12 years old or older,the method comprising administering once daily a pharmaceuticalcomposition comprising 20 mg of substantially pure rofecoxib or 20 ng ofhighly pure rofecoxib, wherein the treatment achieves a reduction of atleast 1 from baseline in a Pain intensity Numerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprising:determining whether the subject has a history or current symptoms ofcardiovascular disease; administering once daily to the subject apharmaceutical composition comprising at least 12.5 mg substantiallypure rofecoxib if it is determined that the subject does not have ahistory or current symptoms of cardiovascular disease, wherein thetreatment achieves a reduction of at least 1 from baseline in a PainIntensity Numerical Rating Scale within 1 week of first administeringthe pharmaceutical composition to the subject.

In some embodiments, the pharmaceutical composition comprises 1 mg, 2mg, 3 mg, 5 mg, 6.25 mg, 7.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg,12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg,17 mg, 17.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22.5 mg, 25 mg, 30 mg,35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, or 70 mg ofsubstantially pure rofecoxib.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of highly pure rofecoxib, wherein thetreatment achieves a greater reduction in Pain Intensity NumericalRating Scale compared to the administering of the same amount of apharmaceutical composition comprising rofecoxib that is not highly pure.

In some embodiments, the pain is caused by one or more conditionsselected from the group comprising of hemophilic arthropathy,osteoarthritis, rheumatoid arthritis, pauciarticular or polyarticularcourse Juvenile Rheumatoid Arthritis (JRA), juvenile idiopathicarthritis, including systemic juvenile idiopathic arthritis, acute pain,primary dysmenorrhea, migraine attacks, or migraine associated with vonWillebrand disease. In other embodiments, the pain is caused bypsoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising an effective amount of highly purerofecoxib, wherein the treatment results in fewer side effects comparedto administering the same amount of a pharmaceutical compositioncomprising of rofecoxib that is not highly pure. In some embodiments,the pain, fever, or inflammation is caused by one or more conditionsselected from the group comprising of hemophilic arthropathy,osteoarthritis, rheumatoid arthritis, pauciarticular or polyarticularcourse Juvenile Rheumatoid Arthritis (JRA), juvenile idiopathicarthritis, acute pain, primary dysmenorrhea, migraine attacks, ormigraine associated with von Willebrand disease. In other embodiments,the pain or inflammation is caused by psoriatic arthritis orfibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever or inflammation caused by one or moreconditions, the method comprising administering to a subject in needthereof a pharmaceutical composition comprising an effective amount ofsubstantially pure rofecoxib and a pharmaceutically acceptable carrieror highly pure mfecoxib and a pharmaceutically acceptable carrierwherein the treatment results in a reduction in pain, fever, orinflammation, further wherein the treatment results in reduction ofeffects associated with administration of rofecoxib that is not highlypure.

In some embodiments, the one or more conditions is selected from thegroup comprising of hemophilic arthropathy, osteoarthritis, rheumatoidarthritis, pauciarticular or polyarticular course Juvenile RheumatoidArthritis (JRA), acute pain, primary dysmenorrhea, migraine attacks, ormigraine associated with von Willebrand disease. In other embodiments,the one or more conditions is psoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of manufacturing substantially pure rofecoxib or highly purerofecoxib as set forth in FIG. 1 , the method comprising the steps of:bromination of 4′-(methylthio)acetophenone (RSM1) in the presence ofTBABr₃ to obtain an intermediate RXB-Bromocetone product at stage 1;involving the intermediate product with Phenylacetic acid (RSM2) in anucleophilic substitution reaction in the presence of sodium hydroxideto obtain a RXB-Phenylacetate product at stage 2; crystallizing theproduct of stage 2 in isopropanol and achieving filterable crystals;intramolecularly cyclizing of the product of stage 2 at 70° C. in DMSOin presence of diisopropylamine; converting4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone to highly purerofecoxib or substantially pure rofecoxib by use of hydrogen peroxidewith a catalytic amount of dihydrate sodium tungsten in acetonitrile;and recrystallizing the rofecoxib in a mixture of DMSO and water.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising rofecoxib comprising less than0.05% of 4-[4-(methylsulfonyl) phenyl]-3-phenyl-2,5-furandione and apharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising highly pure, or substantiallypure, rofecoxib having at least one impurity that is a prodrug ofrofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the rofecoxib is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the rofecoxib comprises less than 0.02%4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides amethod for treating pain, fever, or inflammation in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition comprising an effective amount of rofecoxib comprising lessthan 0.05% 4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione and apharmaceutically acceptable carrier, wherein the subject is within asubject population having a reduced risk of a serious cardiovascularthrombotic event.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. Insome embodiments, the effective amount of rofecoxib is 17.5 mg. In someembodiments, the effective amount of rofecoxib is 20 mg. In someembodiments, the rofecoxib comprises less than 0.02%4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides amethod for treating pain, fever, or inflammation in a subject, whilereducing one or more side effects associated with the administration of4-[4-(methylsulfonyl) phenyl]-3-phenyl-2,5-furandione, comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of rofecoxib comprising less than 0.05% of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione and apharmaceutically acceptable carrier.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. Insome embodiments, the effective amount of rofecoxib is 17.5 mg. In someembodiments, the effective amount of rofecoxib is 20 mg. In someembodiments, the rofecoxib comprises less than 0.05%4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 12.5 mg highly pure rofecoxib oran acceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 17.5 mg highly pure rofecoxib oran acceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 20 mg highly pure rofecoxib or anacceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 12.5 mg substantially purerofecoxib or an acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 17.5 mg substantially purerofecoxib or an acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 20 mg substantially pure rofecoxibor an acceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering an effective amount ofhighly pure rofecoxib.

In some embodiments, the effective amount of highly pure rofecoxib is12.5 mg. In some embodiments, the effective amount of highly purerofecoxib is 17.5 mg. In some embodiments, the effective amount ofhighly pure rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering an effective amount ofsubstantially pure rofecoxib.

In some embodiments, the effective amount of substantially purerofecoxib is 12.5 mg. In some embodiments, the effective amount ofsubstantially pure rofecoxib is 17.5 mg. In some embodiments, theeffective amount of substantially pure rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising an effective amount of rofecoxibhaving less than 0.10%, 0.05%, 0.02%, or 0.01%4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione, and apharmaceutically acceptable carrier.

In some embodiments, the effective amount of rofecoxib is 17.5 mg. Insome embodiments, the effective amount of rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising an effective amount of rofecoxibhaving less than 0.10%, 0.05%, 0.02%, or 0.01%4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one, and apharmaceutically acceptable carrier.

In some embodiments, the effective amount is 10 mg. In some embodiments,the effective amount is 12.5 mg. In some embodiments, the effectiveamount of rofecoxib is 17.5 mg. In some embodiments, the effectiveamount of rofecoxib is 20 mg. In some embodiments, the effective amountof rofecoxib is 25 mg.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a flow diagram for rofecoxib manufacturing process A₁,which avoids production of one or more of the impurities found inpreviously available rofecoxib bulk drug product:4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one; and4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

FIG. 2 shows that after a recrystallization step rofecoxib is about99.9% pure, with about 0.1% or less total impurities. Rofecoxib preparedaccording to the subject matter described herein is also known by thecompound identifier TRM-201 or RXB-201.

FIG. 3 shows representative chromatograms of a mixture that contains 1mg/mL rofecoxib, 1.5 μg/mL4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, and 1.5 μg/mL4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and the blank solution.

FIG. 4 shows the mass spectrum of rofecoxib in acetonitrile.

FIGS. 5A-D show the Nuclear Magnetic Resonance (NMR) spectroscopyanalysis of rofecoxib. FIG. 5A shows the 600 MHz ¹H-NMR spectrum ofrofecoxib in DMSO-d₆. FIG. 5B shows the expanded 600 MHz ¹H-NMR spectrumof rofecoxib in DMSO-d₆. FIG. 5C shows the 125 MHz ¹³C-NMR spectrum ofrofecoxib in DMSO-d₆. FIG. 5D shows the expanded 125 MHz ¹³C-NMRspectrum of rofecoxib in DMSO-d₆.

FIGS. 6A-C show the 2-dimension spectra of rofecoxib. FIG. 6A shows theH-H COSY NMR spectrum of rofecoxib in DMSO-d₆. FIG. 6B shows the HSQCmultiplicity edited NMR Spectrum of rofecoxib in DMSO-d₆. FIG. 6C showsthe HMBC NMR spectrum of rofecoxib in DMSO-d₆.

FIG. 7 shows the infrared (1R) absorbance spectrum of solid rofecoxibacquired using an Attenuated Total Reflectance (ATR) cell.

FIG. 8 shows the single crystal structure of rofecoxib in the CambridgeStructural Database.

FIG. 9 shows an overlay plot showing that the X-ray powder diffraction(XRPD) patterns of the rofecoxib reference standard compares favorablyto the XRPD pattern that was calculated from the crystal structurereported in the Cambridge Structural Database.

FIGS. 10A-B show rofecoxib characteristics. FIG. 10A shows differentialscanning calorimetry (DSC) analysis of rofecoxib. FIG. 10B shows athermogravimetric analysis (TGA) of rofecoxib.

FIG. 11 shows a solubilization and nucleation curve of rofecoxib inDMSO.

FIG. 12 shows solubilization and nucleation curves of rofecoxib in DMSO.

FIG. 13 shows UHPLC results of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation in biphasicconditions.

FIG. 14 shows UHPLC results of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation inacetonitrile/sulfolane mixtures.

FIGS. 15A-B show 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanoneoxidation follow-up. FIG. 15A shows4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation follow-up forCHG P059-074. FIG. 15B shows4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation follow-up forCHG P059-078.

FIG. 16 shows the proposed structure of the4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one(RXB-Hydroxy) impurity.

FIG. 17 shows a general scheme of the oxidation reaction.

FIG. 18 shows a focus on H₂O₂ addition.

FIG. 19 shows maximum temperature attainable by the synthesis reaction.

FIG. 20 shows conversion to product.

FIG. 21 shows oxidation reaction power.

FIG. 22 shows reaction classification system.

FIG. 23 shows a summary of in silico mutagenicity findings.

FIG. 24 shows an optimized oxidation process.

FIG. 25 shows a recrystallization flowsheet of rofecoxib (RXB-201).

FIG. 26 shows a process flowsheet for4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation.

FIG. 27 shows a flowsheet of optimized recrystallization of rofecoxib(RXB-201).

FIG. 28 shows the current flowsheet for4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (RXB-furanone)oxidation.

FIG. 29 shows an optimized flowsheet of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation.

FIG. 30 shows a flowsheet for the calorimetric test.

DETAILED DESCRIPTION Definitions

The following are definitions of terms used in the presentspecification. The initial definition provided for a group or termherein applies to that group or term throughout the presentspecification individually or as part of another group, unless otherwiseindicated. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art.

As used herein, “rofecoxib” refers to the active ingredient4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone or apharmaceutically acceptable salt or solvate thereof. Rofecoxib, and amethod of manufacturing rofecoxib, are described in U.S. Pat. No.5,474,995, which is incorporated herein by reference in its entirety.The rofecoxib as provided herein is produced consistent with andaccording to GMP requirements and is suitable for use in humans. Therofecoxib as described herein may be in amorphous or crystalline form.

The purity of rofecoxib resulting from the manufacturing process asdescribed herein is determined as a percent (%) area basis, typically asquantified by analytical chromatography, such as using HPLC, UHPLC orUPLC.

In some embodiments, the highly pure rofecoxib, resulting from themanufacturing process as described herein, comprises less than or equalto about 0.10%, 0.075%, 0.050%, 0.025%, 0.020%, or 0.001% area basistotal impurities. In some embodiments, the highly pure rofecoxib isessentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the highly pure rofecoxib is essentially free of, or freeof, 4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one. In someembodiments, the highly pure rofecoxib comprises less than or equal toabout 0.10%, 0.05%, 0.02%, or 0.01% area basis of4-[4-(methylthio)phenyl)-3-phenyl-2(5H)-furanone. In some embodiments,the highly pure rofecoxib comprises less than or equal to about 0.10%,0.05%, 0.02%, or 0.01% area basis of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

As used herein, “co-administration” means the administration of twoagents (e.g. concomitantly or sequentially) in any manner in which thepharmacological effects of both are manifest in the subject at the sametime. Concomitant administration does not require that both agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of both agentsneed not manifest themselves at the same time. The effects need only beoverlapping for a period of time and need not be coextensive.

As used herein, “essentially free” means, with respect to an impurity,having less than about 0.10% area basis of the impurity.

As used herein, “free of” means, with respect to an impurity having anamount of the impurity that is below the limitation of detection i.e.less than 0.02% area basis of the impurity.

As used herein, “highly pure” means, with respect to an activeingredient, having less than or equal to about 0.10% area basis totalimpurities.

As used herein, “substantially free” means, with respect to an impurity,having less than or equal to about 0.50% area basis of the impurity.

As used herein, “substantially pure” means, with respect to an activeingredient, having less than or equal to about 0.50% area basis totalimpurities.

As used herein, “limit of detection” means, with respect to an impurity,having at least 0.02% area basis of the impurity.

Prodrugs and solvates of the compounds of the present subject matter arealso contemplated herein. The term “prodrug” as employed herein denotesa compound that, upon administration to a subject, undergoes chemicalconversion by metabolic or chemical processes to yield a compound of thepresent subject matter, or a salt and/or solvate thereof. Solvates ofthe compounds of the present subject matter include, for example,hydrates.

As used herein, “effective amount” refers to any amount that isnecessary or sufficient for achieving or promoting a desired outcome. Insome instances, an effective amount is a therapeutically effectiveamount. A therapeutically effective amount is any amount that isnecessary or sufficient for promoting or achieving a desired biologicalresponse in a subject. The effective amount for any particularapplication can vary depending on such factors as the disease orcondition being treated, the particular agent being administered, thesize of the subject, or the severity of the disease or condition. One ofordinary skill in the art can empirically determine the effective amountof a particular agent without necessitating undue experimentation.

As used herein, the term “subject” refers to a vertebrate animal. In oneembodiment, the subject is a mammal or a mammalian species. In oneembodiment, the subject is a human. In other embodiments, the subject isa non-human vertebrate animal, including, without limitation, non-humanprimates, laboratory animals, livestock, racehorses, domesticatedanimals, and non-domesticated animals.

As used herein, the term “patient” refers to a human or animal.

The term “mammal” includes, but is not limited to, a human, mouse, rat,guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as amonkey, chimpanzee, baboon or rhesus. In one embodiment, the mammal is ahuman.

As used herein, “gastro-protective agent” includes, but is not limitedto, an antacid therapy, a proton pump inhibitor, a H2 receptorantagonist, or misoprostol.

As used herein, “bleeding disorder” includes, but is not limited to,hemophilia A (factor VIII deficiency), hemophilia B (factor IXdeficiency), von Willebrand disease, rare factor deficiencies includingI, II, V, VII, X, XI, XII and XIII, and drug-induced bleeding disorders.

Compositions of the Present Subject Matter

Rofecoxib (also known as4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone) is a nonsteroidalanti-inflammatory drug that exhibits anti-inflammatory, analgesic, andantipyretic activities. Without being bound by theory, the mechanism ofaction of rofecoxib is believed to be due to inhibition of prostaglandinsynthesis, via inhibition of cyclooxygenase-2 (COX-2). Additionally, attherapeutic concentrations in humans, rofecoxib does not inhibit thecyclooxygenase-1 (COX-1) isoenzyme. The chemical structure of rofecoxibis shown below. Rofecoxib bears no chiral centers and has a molecularweight of 314.355 g moL⁻¹.

Despite being withdrawn from the market many years ago, and consideredunsafe for human use, a surprising discovery shows that rofecoxib issafe to treat a number of conditions and diseases. For example, there isa long felt but unmet need for new, non-opioid based pain medicationsfor subjects suffering from pain that is co-morbid or associated withdiseases or conditions arising from bleeding disorders, including butnot limited to hemophilic arthropathy and von Willebrand's disease.

In addition to discovering that rofecoxib may be used to safely treat anumber of diseases, it has been surprisingly discovered that the safetyand efficacy profile of rofecoxib may be enhanced by administering apharmaceutical composition comprising rofecoxib as described herein thatis substantially pure or highly pure, or essentially free or free of oneor more impurities found in previously available rofecoxib drug product.

Prior methods of manufacturing rofecoxib produced rofecoxib drugsubstance containing certain impurities, some of which have been linkedto the safety concerns that prompted the withdrawal of the previouslyavailable “VIOXX” product from the market in 2004. Without being boundby theory, oxidation of the conjugate base of rofecoxib is one processthought to introduce impurities, which upon entry into a subject'ssystem may survive long enough to react with nucleophilic groups ofbiomolecules, tissues and amino groups. Therefore, it is thought thatthese impurities may lead to a low-level chronic toxicity that iscumulative and dangerous over periods of many months. It is thought thatfor this reason the cardiotoxicity of VIOXX was not readily observedduring short-term (one year or less) studies. The impurities thought toresult from oxidation of rofecoxib include4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. See Reddy et al.,Facile air oxidation of the conjugate base of rofecoxib (Vioxx), apossible contributor to chronic human toxicity. Tetrahedron Lett 46:927-929 (2005).

The novel manufacturing process described herein surprisingly produceshigh yields of substantially pure or highly pure rofecoxib, or rofecoxibessentially free or free of those undesirable impurities. As furtherdescribed herein, it was also surprisingly discovered that this novelmanufacturing process produces one or more prodrugs of rofecoxib havingbeneficial therapeutic properties.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising highly pure rofecoxib or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In some embodiments, the highly pure rofecoxib comprises less than about0.10%, about 0.075%, about 0.05%, about 0.025%, about 0.02%, about 0.01,or about 0.001% total impurities. In some embodiments, the highly purerofecoxib is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the highly pure rofecoxib comprises less than about 0.10%,about 0.05%, about 0.02%, or about 0.01% of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. In some embodiments,the highly pure rofecoxib comprises less than about 0.10%, about 0.05%,about 0.02%, or about 0.01% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising substantially pure rofecoxib or anacceptable salt thereof and a pharmaceutically acceptable carrier.

In some embodiments, the substantially pure rofecoxib comprises lessthan about 0.40%, about 0.30%, about 0.25%, about 0.20%, or about 0.15%total impurities. In some embodiments, the substantially pure rofecoxibis substantially free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the substantially pure rofecoxib comprises less than about0.25%, about 0.20%, or about 0.15% of4-[4-(methylthio)phenyl]-3-phenyl-2(51H)-furanone. In some embodiments,the substantially pure rofecoxib comprises less than about 0.25%, about0.20%, or about 0.15% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

In some embodiments, the highly pure rofecoxib comprises less than about0.10%, about 0.05%, about 0.02%, about 0.01%, or is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one. In someembodiments, the highly pure rofecoxib comprises less than about 0.10%,about 0.05%, about 0.02%, about 0.01%, or is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising an effective amount of highly purerofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the pain, fever, or inflammation is caused by oneor more conditions selected from the group comprising of hemophilicarthropathy, osteoarthritis. rheumatoid arthritis, pauciarticular orpolyarticular course Juvenile Rheumatoid Arthritis (JRA), juvenileidiopathic arthritis, acute pain, primary dysmenorrhea, migraineattacks, or migraine associated with von Willebrand disease. In otherembodiments, the pain or inflammation is caused by psoriatic arthritisor fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising an effective amount ofsubstantially pure rofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the pain, fever, or inflammation is caused by oneor more conditions selected from the group comprising of hemophilicarthropathy, osteoarthritis, rheumatoid arthritis, pauciarticular orpolyarticular course Juvenile Rheumatoid Arthritis (JRA), juvenileidiopathic arthritis, acute pain, primary dysmenorrhea, migraineattacks, or migraine associated with von Willebrand disease. In otherembodiments, the one or more conditions is psoriatic arthritis orfibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain or migraine associated with a condition causedby a bleeding disorder in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of highly pure rofecoxib and apharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain or migraine associated with a condition causedby a bleeding disorder in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of substantially pure rofecoxib and apharmaceutically acceptable carrier.

In some embodiments, the subject is 2 years of age or older. In someembodiments, the subject is 12 years old or older. In some embodiments,the subject is 12 years old to 75 years old. In some embodiments, thesubject does not have a history or current symptoms of cardiovasculardisease. In some embodiments, the subject does not have a history orcurrent symptoms of gastrointestinal bleeding, ulceration, orperforation. In some embodiments, the pharmaceutical composition isadministered once daily. In some embodiments, the pharmaceuticalcomposition is administered two times or more daily. In someembodiments, the condition is caused by a bleeding disorder. In someembodiments, the bleeding disorder is hemophilia A or B, C vonWillebrand Disease, or a drug-induced bleeding disorder. In someembodiments, the condition is hemophilic arthropathy, juvenileidiopathic arthritis, or migraine associated with von Willebranddisease.

In some embodiments, the treatment is effective at treating pain withoutco-administration of an analgesic. In some embodiments, the treatmentresults in a subject decreasing or discontinuing use of analgesics orrescue medications during the treatment as compared to before initiationof the treatment. In some embodiments, the treatment results in asubject decreasing or discontinuing use of opioid medications during thetreatment.

In some embodiments, the method further includes administering agastro-protective agent. In some embodiments, the gastro-protectiveagent is co-administered with the pharmaceutical composition. In someembodiments, the treatment does not include administeringgastro-protective agent.

In some embodiments, the treatment achieves a reduction of at least 1from baseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 2 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 3 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 4 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the treatment achieves a reduction of at least 5 frombaseline in a Pain Intensity Numerical Rating Scale. In someembodiments, the reduction in the Pain Intensity Numerical Rating Scaleis achieved within 1, 2, 3, 4, 5, 6 days, 1 week, or 2 weeks ofadministering the pharmaceutical composition.

In some embodiments, the effective amount of the rofecoxib administeredto a subject is 12.5 mg. In some embodiments, the effective amount ofthe rofecoxib administered to a subject is 17.5 mg. In some embodiments,the effective amount of the rofecoxib administered to a subject is 20mg. In some embodiments, the effective amount of the rofecoxibadministered to a subject is 25 mg.

In some embodiments, the effective amount of the rofecoxib administeredto a subject is selected from the group comprising of 1 mg, 2 mg, 3 mg,5 mg, 6.25 mg, 7.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg,13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 ng, 15.5 mg, 16 mg, 16.5 mg, 17 mg,17.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22.5 mg, 25 mg, 30 mg, 35 mg,40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, and 70 mg. In someembodiments, the effective amount of the rofecoxib administered to asubject is selected from the group comprising of 0.10 mg/kg, 0.15 mg/kg,0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg,0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, or 0.70 mg/kg.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering once daily to the subject a pharmaceutical compositioncomprising 12.5 mg of substantially pure rofecoxib or 12.5 mg of highlypure rofecoxib as a sole active ingredient, wherein the treatmentachieves a reduction of at least 1 from baseline in a Pain IntensityNumerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering once daily to the subject a pharmaceutical compositioncomprising 17.5 mg of substantially pure rofecoxib or 17.5 mg of highlypure rofecoxib as a sole active ingredient, wherein the treatmentachieves a reduction of at least 1 from baseline in a Pain IntensityNumerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering once daily to the subject a pharmaceutical compositioncomprising 20 mg of substantially pure rofecoxib or 20 mg of highly purerofecoxib as a sole active ingredient, wherein the treatment achieves areduction of at least 1 from baseline in a Pain Intensity NumericalRating Scale.

In some embodiments, the pharmaceutical composition achieves a reductionin a Pain Intensity Numerical Rating Scale that is equal to or greaterthan the once daily administration of a pharmaceutical compositioncomprising 25 mg of rofecoxib that is not highly pure. In someembodiments, the condition is hemophilic arthropathy.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with hemophilic arthropathy in asubject in need thereof, wherein the subject is 12 years old or older,the method comprising administering once daily a pharmaceuticalcomposition comprising 12.5 mg of substantially pure rofecoxib or 12.5mg of highly pure rofecoxib, wherein the treatment achieves a reductionof at least 1 from baseline in a Pain Intensity Numerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with hemophilic arthropathy in asubject in need thereof, wherein the subject is 12 years old or older,the method comprising administering once daily a pharmaceuticalcomposition comprising 17.5 mg of substantially pure rofecoxib or 17.5mg of highly pure rofecoxib, wherein the treatment achieves a reductionof at least 1 from baseline in a Pain Intensity Numerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with hemophilic arthropathy in asubject in need thereof, wherein the subject is 12 years old or older,the method comprising administering once daily a pharmaceuticalcomposition comprising 20 mg of substantially pure rofecoxib or 20 mg ofhighly pure rofecoxib, wherein the treatment achieves a reduction of atleast 1 from baseline in a Pain Intensity Numerical Rating Scale.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprising:determining whether the subject has a history or current symptoms ofcardiovascular disease; administering once daily to the subject apharmaceutical composition comprising at least 12.5 mg substantiallypure rofecoxib if it is determined that the subject does not have ahistory or current symptoms of cardiovascular disease, wherein thetreatment achieves a reduction of at least 1 from baseline in a PainIntensity Numerical Rating Scale within 1 week of first administeringthe pharmaceutical composition to the subject.

In some embodiments, the pharmaceutical composition comprises 1 mg, 2mg, 3 mg, 5 mg, 6.25 mg, 7.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg,12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg,17 mg, 17.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22.5 mg, 25 mg, 30 mg,35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, or 70 mg ofsubstantially pure rofecoxib.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of highly pure rofecoxib, wherein thetreatment achieves a greater reduction in Pain Intensity NumericalRating Scale compared to the administering of the same amount of apharmaceutical composition comprising rofecoxib that is not highly pure.

In some embodiments, the pain is caused by one or more conditionsselected from the group comprising of hemophilic arthropathy,osteoarthritis, rheumatoid arthritis, pauciarticular or polyarticularcourse Juvenile Rheumatoid Arthritis (JRA), juvenile idiopathicarthritis, including systemic juvenile idiopathic arthritis, acute pain,primary dysmenorrhea, migraine attacks, or migraine associated with vonWillebrand disease. In other embodiments, the pain is caused bypsoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering to the subject apharmaceutical composition comprising an effective amount of highly purerofecoxib, wherein the treatment results in fewer side effects comparedto administering the same amount of a pharmaceutical compositioncomprising of rofecoxib that is not highly pure. In some embodiments,the pain, fever, or inflammation is caused by one or more conditionsselected from the group comprising of hemophilic arthropathy,osteoarthritis, rheumatoid arthritis, pauciarticular or polyarticularcourse Juvenile Rheumatoid Arthritis (JRA), juvenile idiopathicarthritis, acute pain, primary dysmenorrhea, migraine attacks, ormigraine associated with von Willebrand disease. In other embodiments,the pain or inflammation is caused by psoriatic arthritis orfibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever or inflammation caused by one or moreconditions, the method comprising administering to a subject in needthereof a pharmaceutical composition comprising an effective amount ofsubstantially pure rofecoxib and a pharmaceutically acceptable carrieror highly pure rofecoxib and a pharmaceutically acceptable carrierwherein the treatment results in a reduction in pain, further whereinthe treatment results in reduction of effects associated withadministration of rofecoxib that is not highly pure.

In some embodiments, the one or more conditions is selected from thegroup comprising of hemophilic arthropathy, osteoarthritis, rheumatoidarthritis, pauciarticular or polyarticular course Juvenile RheumatoidArthritis (JRA), acute pain, primary dysmenorrhea, migraine attacks, ormigraine associated with von Willebrand disease. In other embodiments,the one or more conditions is psoriatic arthritis or fibromyalgia.

In certain aspects, the subject matter disclosed herein provides amethod of manufacturing substantially pure rofecoxib or highly purerofecoxib as set forth in FIG. 1 , the method comprising the steps of:bromination of 4′-(methylthio)acetophenone (RSM1) in the presence ofTBABr₃ to obtain an intermediate RXB-Bromocetone product at stage 1;involving the intermediate product with Phenylacetic acid (RSM2) in anucleophilic substitution reaction in the presence of sodium hydroxideto obtain a RXB-Phenylacetate product at stage 2; crystallizing theproduct of stage 2 in isopropanol and achieving filterable crystals;intramolecularly cyclizing of the product of stage 2 at 70° C. in DMSOin presence of diisopropylamine; converting4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone to highly purerofecoxib or substantially pure rofecoxib by use of hydrogen peroxidewith a catalytic amount of dihydrate sodium tungsten in acetonitrile;and recrystallizing the rofecoxib in a mixture of DMSO and water.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising rofecoxib comprising less than0.05% of 4-[4-(methylsulfonyl) phenyl]-3-phenyl-2,5-furandione and apharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising highly pure, or substantiallypure, rofecoxib having at least one impurity that is a prodrug ofrofecoxib and a pharmaceutically acceptable carrier.

In some embodiments, the rofecoxib is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. In someembodiments, the rofecoxib comprises less than 0.02%4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides amethod for treating pain, fever, or inflammation in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition comprising an effective amount of rofecoxib comprising lessthan 0.05% 4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione and apharmaceutically acceptable carrier, wherein the subject is within asubject population having a reduced risk of a serious cardiovascularthrombotic event.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. Insome embodiments, the effective amount of rofecoxib is 17.5 mg. In someembodiments, the effective amount of rofecoxib is 20 mg. In someembodiments, the rofecoxib comprises less than 0.02%4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides amethod for treating pain, fever, or inflammation in a subject, whilereducing one or more side effects associated with the administration of4-[4-(methylsulfonyl) phenyl]-3-phenyl-2,5-furandione, comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of rofecoxib comprising less than 0.05% of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione and apharmaceutically acceptable carrier.

In some embodiments, the effective amount of rofecoxib is 12.5 mg. Insome embodiments, the effective amount of rofecoxib is 17.5 mg. In someembodiments, the effective amount of rofecoxib is 20 mg. In someembodiments, the rofecoxib comprises less than 0.05%4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 12.5 mg highly pure rofecoxib oran acceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 17.5 mg highly pure rofecoxib oran acceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 20 mg highly pure rofecoxib or anacceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 12.5 mg substantially purerofecoxib or an acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 17.5 mg substantially purerofecoxib or an acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising 20 mg substantially pure rofecoxibor an acceptable salt thereof and a pharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering an effective amount ofhighly pure rofecoxib.

In some embodiments, the effective amount of highly pure rofecoxib is12.5 mg. In some embodiments, the effective amount of highly purerofecoxib is 17.5 mg. In some embodiments, the effective amount ofhighly pure rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain, fever, or inflammation in a subject in needthereof, the method comprising administering an effective amount ofsubstantially pure rofecoxib.

In some embodiments, the effective amount of substantially purerofecoxib is 12.5 mg. In some embodiments, the effective amount ofsubstantially pure rofecoxib is 17.5 mg. In some embodiments, theeffective amount of substantially pure rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising an effective amount of rofecoxibhaving less than 0.10%, 0.05%, 0.02%, or 0.01%4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione, and apharmaceutically acceptable carrier.

In some embodiments, the effective amount of rofecoxib is 17.5 mg. Insome embodiments, the effective amount of rofecoxib is 20 mg.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising an effective amount of rofecoxibhaving less than 0.10%, 0.05%, 0.02%. or 0.01%4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one, and apharmaceutically acceptable carrier.

In some embodiments, the effective amount is 10 mg. In some embodiments,the effective amount is 12.5 mg. In some embodiments, the effectiveamount of rofecoxib is 17.5 mg. In some embodiments, the effectiveamount of rofecoxib is 20 mg. In some embodiments, the effective amountof rofecoxib is 25 mg.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising rofecoxib or an acceptable saltthereof that is substantially free of one or mom of the impuritiesselected from the group consisting of:4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone, and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, and apharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides apharmaceutical composition comprising rofecoxib or an acceptable saltthereof that is essentially free of one or more impurities selected fromthe group consisting of:4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone, and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, and apharmaceutically acceptable carrier.

In some aspects, the subject matter disclosed herein provides a methodof treating pain, fever, or inflammation caused by one or moreconditions in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprisingrofecoxib or an acceptable salt thereof that is essentially free of oneor more impurities selected from the group consisting of:4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone, and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, and apharmaceutically acceptable carrier.

In certain aspects, the subject matter disclosed herein provides amethod of treating pain associated with a condition caused by a bleedingdisorder in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of rofecoxib or an acceptable salt thereof that isessentially free of one or more impurities selected from the groupconsisting of: 4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone, and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, and apharmaceutically acceptable carrier.

The subject matter disclosed herein relates to substantially pure orhighly pure rofecoxib, also known as TRM-201 or RXB-201, or apharmaceutically acceptable salt or solvate thereof, having a favorableimpurity profile, rofecoxib method of manufacture, and the use ofpharmaceutical compositions containing rofecoxib to treat or preventvarious conditions and diseases.

In one embodiment, the rofecoxib as provided herein is, with respect toall impurities, substantially pure or highly pure. In anotherembodiment, the rofecoxib as provided herein is substantially free of,essentially free of, or free of,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. In one aspect, therofecoxib as provided herein is substantially free of, essentially freeof, or free of, 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

As described herein, it has been found that rofecoxib, althoughpreviously considered to be unsafe for human use, may be manufactured ina substantially pure or highly pure form, substantially free,essentially free, or free of impurities including those found inpreviously available rofecoxib bulk drug product, and may be safelyadministered to humans as the active ingredient in a pharmaceuticalcomposition for a number of diseases or conditions including, but notlimited to, diseases or conditions arising from or comorbid withbleeding disorders, such as hemophilic arthropathy. In one aspect, thesubject matter described herein addresses the long felt. but unmet need,for new treatments for hemophilic arthropathy through the safeadministration of rofecoxib or a pharmaceutically acceptable salt orsolvate thereof.

In another aspect, the subject matter described herein relates totreatments for juvenile idiopathic arthritis, including systemicjuvenile idiopathic arthritis (SJIA) through the safe administration ofrofecoxib or a pharmaceutically acceptable salt or solvate thereof. Inyet another aspect, the subject matter described herein relates totreatments for migraine associated with von Willebrand disease throughthe safe administration of rofecoxib or a pharmaceutically acceptablesalt or solvate thereof, wherein the subjects who are treated expressvon Willebrand factor at a level about 50% below normal.

The subject matter disclosed herein includes, but is not limited to, thetreatment of various diseases or conditions through the administrationof a pharmaceutical composition comprising rofecoxib or apharmaceutically acceptable salt or solvate thereof, which isessentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione, wherein thetreatment results in greater efficacy and/or reduced side effects,including those reported as adverse events or serious adverse events,compared to previously available rofecoxib bulk drug product, thusfacilitating safe, long term use of the pharmaceutical composition.

Purity of the rofecoxib resulting from the manufacturing processdescribed herein is determined as a percent (%) area basis, typically asquantified by analytical chromatography, such as using HPLC, UHPLC, UPLCor other analytical means in the art.

Manufacture of Rofecoxib

In certain aspects, the rofecoxib as provided herein is manufactured ina manner that produces rofecoxib that is essentially free of, or freeof, 4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione, both of which areimpurities found in previously available rofecoxib bulk drug product.

In one aspect, rofecoxib as provided herein may be manufacturedaccording to a process as shown in FIG. 1 . In one aspect, the processrelies on two starting materials. In one aspect, rofecoxib ismanufactured via a four-step sequential process followed by acrystallization step and a micronization step. The first step mayinclude the bromination of 4′-(Methylthio)acetophenone (SM1) in thepresence of TBABr₃, leading to the formation of intermediate step 1,RXB-Bromoacetone. In one embodiment, 4′-(Methylthio)acetophenone isdissolved in a methanol and dichloromethane mixture to whichtetrabutylammonium tribromide (TBAB₃) dissolved in dichloromethane isadded to form RXB-Bromocetone. Upon completion, water can be added toquench the reaction. The layers can then be separated, and the waterlayer decanted. In one embodiment, the RXB-Bromocetone is not isolated.

In one embodiment, the bromination reaction of step one is followed byan esterification reaction in step two of the rofecoxib manufacturingprocess. In step two, phenylacetic acid and sodium hydroxide can bedissolved in water. The solution of phenylacetate can then be added tothe RXB-Bromocetone solution from step one to form the step 2 product,RXB-Phenylacetate. At step two, there may be distillation andcrystallization in isopropanol. The solvent volume can be reduced bydistillation at about 55° C. under atmospheric pressure and isopropanolcan be added to the mixture, which is then cooled to about 0° C. toyield solid RXB-Phenylacetate. Seed crystals may be added during thecooling process. In one embodiment, the RXB-Phenylacetate is isolatedfrom the resulting slurry by centrifugation at about 0° C., and thecollected solids are then washed with isopropanol. The resulting filtercake can be dried under vacuum at about 60° C.

In one embodiment, step three of the rofecoxib manufacturing process asshown in FIG. 1 involves preparation of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (referred to in FIG. 1as “RXB-furanone”). In one embodiment. RXB-Phenylacetate is indimethylsulfoxide (DMSO) and heated to about 70° C. To this solution,diisopropylamine (DIPA) and additional DMSO can be added and theresulting solution can be maintained at temperature until the reactionis complete. Isopropanol can be added to the mixture, which can thencooled to about 0° C. to yield solid4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (referred to in FIG. 1as “RXB-furanone”). Seed crystals may be added during the coolingprocess. The 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (referredto in FIG. 1 as “RXB-furanone”) can be isolated from the resultingslurry by centrifugation at about 0° C., and the collected solids canthen be washed with isopropanol. In one embodiment, the isolated4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (referred to in FIG. 1as “RXB-furanone”) is not dried.

In one embodiment, step four of the process involves preparation ofrofecoxib. In one embodiment, step four involves sulfoxide formation. Instep four of the process,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (referred to in FIG. 1as “RXB-furanone”) and a catalytic amount of sodium tungstate dihydratecan be suspended in acetonitrile and heated to about 65° C. To thissuspension, aqueous hydrogen peroxide can be added. In one embodiment.the resulting slurry is maintained at temperature until the reaction iscomplete, then it is allowed to cool to ambient temperature. Aqueoussodium sulfite solution can be added to the slurry and mixed. In oneembodiment, the crude rofecoxib is isolated from the slurry bycentrifugation at about 0° C., and the collected solids are then washedwith water and isopropanol. In one embodiment, the isolated rofecoxib isnot dried.

In one embodiment, step five involves crystallization of rofecoxib.Crude rofecoxib can be dissolved in DMSO and filtered at about 40° C.The filtrate can then be heated to about 50° C., and purified water canbe added to induce crystallization. The suspension can then be cooledslowly to about 20° C. The resulting slurry can be filtered, and thecollected solids can be washed with a DMSO-water mixture, water, andisopropanol. In one embodiment, the resulting filter cake is dried atabout 60° C. under vacuum. In one embodiment, step six of the processincludes micronization of crystalized rofecoxib using a jet mill. Insome embodiments, the d90 particle size of the rofecoxib followingmicronization is less than about 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, or 4 μm. In some embodiments, the d50particle size of the rofecoxib following micronization is less thanabout 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm. In someembodiments, the d10 particle size of the rofecoxib followingmicronization is less than about 4 μm, 3 μm, 2 μm, 1 μm, 0.9 μm, 0.8 μm,0.7 μm, 0.6 μm, or 0.5 μm. In some embodiments, the particle sizedistribution of the rofecoxib following micronization is as follows: a)the d90 particle size is about 10-12 μm; b) the d50 particle size isabout 3-4 μm; and c) the d10 particle size is about 0.5-1.0 μm.

In accordance with the process shown in FIG. 1 , the final intermediate,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (CAS Number162012-30-8) (referred to in FIG. 2 as “RXB-furanone”), can be convertedto rofecoxib through an oxidation step, for example, by use of anoxidizing agent. In one aspect, the oxidizing agent is hydrogen peroxidewith a catalytic amount of dihydrate sodium tungsten in acetonitrile. Inanother aspect, the oxidizing agent is not potassium peroxymonosulfate(oxone). The oxidation reaction can transform the sulfide function ofstep 3 into the corresponding sulfone using hydrogen peroxide with acatalytic amount of dihydrate sodium tungsten in acetonitrile. It wassurprisingly discovered that increasing the reaction temperature to atleast 50° C., preferably to at least 60° C., and more preferably to 65°C. as well as increasing the amount of solvent (acetonitrile), candecrease the amount of impurities that resulted in substantially purerofecoxib prior to any recrystallization step. In one aspect, theoxidation step may produce rofecoxib that is at least 99.7% pure, i.e.contains less than 0.30% impurities, prior to any recrystallizationstep. In another aspect, the oxidation step produces rofecoxib thatcontains less than 0.15% or 0.10%4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone prior to anyrecrystallization step. In one aspect, the oxidation step producesrofecoxib that contains less than 0.15%, 0.10%, 0.075%, or 0.05%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone (referred to inFIG. 2 as “RXB-Sulfoxide”) prior to any recrystallization step. Inanother aspect, the oxidation step in acetonitrile may produce rofecoxibthat does not require any drying prior to recrystallization.

In one aspect, the oxidation product is recrystallized in a mixture ofDMSO and water. which can remove a pale-yellow coloration and residualimpurities, and can afford the desired active pharmaceutical ingredient,rofecoxib as described herein. It was surprisingly discovered that DMSOmay be used as a solvent in the recrystallization step, producing yieldssimilar to those where dimethyl formamide (DMF) is used as therecrystallization solvent, while avoiding the safety concerns associatedwith use of DMF in the final recrystallization step. In one aspect, therofecoxib as described herein contains no detectable DMF.

It was also surprisingly discovered that the process ofrecrystallization described herein produced rofecoxib essentially free,or free of, 6-methylsulfonylphenanthro-[9,10-C] furan-1(3H)-one, whichhas been described as a photo-cyclization degradation product ofrofecoxib that may be present “without appropriate control of therecrystallisation procedures”. See Dean P M. Structural Examination of6-Methylsulfonylphenanthro-[9,10-C]-furan-1(3H)-one—A RofecoxibDegradation Product. Pharmaceuticals (Basel), 2010; 3(2):369-378.Published 2010 Feb. 1. doi:10.3390/ph3020369.

In one aspect, the process shown in FIG. 1 produces rofecoxib asdescribed herein that is substantially pure or highly pure. In anotheraspect, the rofecoxib produced from the final intermediate isrecrystallized, for example in DMSO and water, to produce substantiallypure or highly pure rofecoxib as described herein.

It was surprisingly found that the oxidation step (Stage 3 to Stage 4)results in the formation of rofecoxib as provided herein that containsonly two impurities at or above the limit of detection,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. In one aspect, theoxidation step results in the formation of rofecoxib that contains lessthan 0.25%, 0.20%, 0.15%, or 0.10%4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and/or4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. In one aspect, theoxidation step results in the formation of rofecoxib as provided hereinthat is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione, or contains eitheror both of those impurities in an amount that is otherwise below thelimit of detection. As a result, the manufacturing process describedherein avoids the production of a compound,4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione, which has beenidentified in the literature as “a possible contributor to chronic humantoxicity”. See Reddy et al., Facile air oxidation of the conjugate baseof rofecoxib (Vioxx), a possible contributor to chronic human toxicity,Tetrahedron Lett 46: 927-929 (2005).

It was also surprisingly discovered that the manufacturing processdescribed herein produced at least one impurity that has beneficialproperties, specifically4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, which is a prodrugof rofecoxib and has been described as having “a slightly improvedpharmacokinetic profile and a better pharmacological activity in anarthritis model . . . when compared with rofecoxib”. See Caturla,Francisco et al., “Racemic and chiral sulfoxides as potential prodrugsof the COX-2 inhibitors Vioxx® and Arcoxia®,” Bioorganic & MedicinalChemistry Letters, 16:3209-3212 (2006).

In one embodiment, evaluation of the potential toxicity of startingmaterials and intermediates is conducted in silica. Rule-based DEREKsoftware using all 51 endpoints, which covers all the major organsystems as well as mutagenicity and skin sensitization, can be used toevaluate each compound. Additionally, for prediction of potentialmutagenicity, the structures can be evaluated using thestatistical-based Leadscope Model Applier. Per ICH M7, use of twocomplementary in silica methods is adequate for the overalldetermination of mutagenicity. The in silica evaluations havedemonstrated that both starting materials and all intermediates in themanufacturing process of rofecoxib are predicted negative formutagenicity in two complementary methods. Therefore, special controlsfor mutagenic compounds does not have to be necessarily employed in themanufacture of rofecoxib.

In one embodiment, acetic acid is an excellent solvent for4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone (>1000 g/L). In oneembodiment, a the starting material may include a spiking of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

The purity of the resulting rofecoxib described as herein is determinedas a percent (%) area basis, typically as quantified by analyticalchromatography, such as using HPLC, UHPLC, UPLC or other analyticalmeans in the art.

Impurity Profile

It was found that the process shown in FIG. 1 can avoid production ofone or more of the impurities found in previously available rofecoxibbulk drug product:4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one; and4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione. See Ahuja et al.,Rofecoxib: an update on physicochemical, pharmaceutical, pharmacodynamicand pharmacokinetic aspects. Journal of Pharmacy and Pharmacology, 2003,55: 859-894.

Additionally, there are two potential impurities that can arise duringthe last bond-forming step (Step 4) of the rofecoxib manufacturingprocess: 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone,4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone is the Step 3 product,and 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is apartially-oxidized intermediate.

FIG. 3 shows representative chromatograms of a mixture that contains 1mg/mL rofecoxib, 1.5 μg/mL4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, and 1.5 μg/mL4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and the blank solution.Table 1 shows that the components of the mixture are well-resolved. Thelimit of quantitation is <0.05% area basis. which is the reportingthreshold. Other potential impurities that may arise during manufactureare residual solvents (acetonitrile, dichloromethane, dimethylsulfoxide,isopropanol, and methanol) and inorganic material.

TABLE 1 Specificity Results for the UHPLC Purity Method Component RT,(min) RRT Rs Unknown impurity 1 1.13 0.45 — RXB-sulfoxide 1.27 0.50  2.0Unknown impurity 2 1.62 0.64  5.7 Rofecoxib 2.52 1.00 11.4 RXB-furanone11.69  4.64 67.4 RT = retention time; RRT = relative retention time;R_(s) = resolution

As a result, in one aspect of the subject matter disclosed herein, therofecoxib incorporated into a pharmaceutical composition may beessentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In addition to reducing or eliminating known impurities in priorformulations of rofecoxib, it was found that the process shown in FIG. 1can produce substantially pure, or highly pure, rofecoxib. Specifically,after the recrystallization step described above, the rofecoxib asdescribed herein can be about 99.9% pure, with about 0.1% or less totalimpurities as detailed in FIG. 2 . It was also found that the processshown in FIG. 1 can produce substantially pure, or highly pure,rofecoxib that contains at least one impurity having beneficialtherapeutic effects that is in an amount above the limit of detection.In one embodiment, the impurity having beneficial therapeutic effects isa prodrug of rofecoxib, such as4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

In one aspect of the subject matter disclosed herein, the substantiallypure rofecoxib contains less than about 0.40%, 0.30%, 0.25%, 0.20%, or0.15% total impurities.

In another aspect, highly pure rofecoxib as provided herein containsless than about 0.075%, 0.050%, 0.025%, 0.020%, or 0.001% totalimpurities.

In one aspect, the rofecoxib as provided herein contains less than about0.25%, 0.20%, 0.15%, 0.10%, 0.05%, 0.02% or 0.01% of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and/or4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone as an impurity. Inother aspects, the rofecoxib as provided herein contains greater than orequal to about 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, or 0.10%. but in allcases less than or equal to about 0.15%. of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and/or4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone as an impurity.

In other aspects, the rofecoxib as provided herein contains less thanabout 0.10%, 0.05%, 0.02%, 0.01%, or is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one. In anotheraspect, the rofecoxib as provided herein contains less than about 0.10%,0.05%, 0.02%, 0.01%, or is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.

In one embodiment, representative sampling by taking and analyzing asuspension in a stirred suspension shows that a full conversion of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone can be reached,wherein 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is notdetected. In one embodiment, sampling 1 mL reaction medium by cooling toroom temperature, filtration and analyzing cake and mother liquors showsthe presence of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone at0.02% area basis in the cake. In one embodiment, sampling 1 mL reactionmedium by cooling to room temperature and addition of 2V water,filtration and analysis of cake and mother liquors shows4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone at 0.02% area basisin the cake. In one embodiment, analysis of the whole product of theexperiment, obtained after cooling the reaction medium to roomtemperature. addition of 2V water, cooling to 0° C., filtration andstandard cake washings, shows4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone content at 0.02%area basis in the cake.

Without being bound by theory, in one embodiment, the presence of abasic salt in the crude or final product, such as sodium sulfite, sodiumsulfide, sodium acetate, sodium bicarbonate, calcium carbonate, orpotassium cyanide is thought to contribute to the production of the4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one impurity. Inone embodiment, control of the rate of production of basic salts in themanufacture of crude rofecoxib, or avoidance of using or forming a basicsalt in the oxidation or recrystallization step, is crucial for theUHPLC profile of the recrystallized compound. Clarifying filtration canbe efficient in avoiding increased4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one levels duringUHPLC analysis. In one embodiment, quench of the basic salt, e.g. sodiumsulfite, sodium sulfide, sodium acetate, sodium bicarbonate, calciumcarbonate, or potassium cyanide, is suppressed in the oxidation processof step 4. In another embodiment, the basic salt may be removed from thecrude or final product by polishing filtration. In yet anotherembodiment, performing the manufacturing process in an inert atmospherealone, or in combination with a basic salt quench, can be efficient inavoiding increased4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one levels duringUHPLC analysis. In some embodiments, performing the manufacturingprocess in an inert atmosphere alone, or in combination with a basicsalt quench, forms rofecoxib substantially free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one levels duringUHPLC analysis. In some embodiments, crude rofecoxib is formed bysulfoxide formation. In some embodiments, crude rofecoxib is formed bysuspending 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and acatalytic amount of sodium tungstate dihydrate are suspended inacetonitrile. In some embodiments, crude rofecoxib is formed by heatingthe 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and tungstatesuspension to about 65° C. In some embodiments, crude rofecoxib isformed by maintaining the slurry at temperature until the reaction iscomplete, then it is allowed to cool to ambient temperature. In someembodiments, crude rofecoxib is formed by adding aqueous sodium sulfitesolution to the slurry. In some embodiments, crude rofecoxib is formedby isolating it from the slurry by centrifugation at 0° C. In someembodiments, crude rofecoxib is obtained by washing the collected solidswith water and isopropanol. In some embodiments. rofecoxib is formed bythe steps of bromination of 4′-(methylthio)acetophenone in the presenceof TBABr3 to obtain an intermediate RXB-Bromocetone product, involvingthe intermediate product with Phenylacetic acid (RSM2) in a nucleophilicsubstitution reaction in the presence of sodium hydroxide to obtain aRXB-Phenylacetate product, crystallizing the product in isopropanol andachieving filterable crystals, intramolecularly cyclizing of the productof stage 2 at 70° C. in DMSO in presence of diisopropylamine, converting4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone to highly purerofecoxib or substantially pure rofecoxib by use of hydrogen peroxidewith a catalytic amount of dihydrate sodium tungsten in acetonitrile,and recrystallizing the rofecoxib in a mixture of DMSO and water.

Purity of the resulting rofecoxib as described herein is determined as apercent area basis, typically as quantified by analyticalchromatography, such as using HPLC, UHPLC, UPLC or other analyticalmeans in the art.

Manufacturing Process Development

Several routes for synthesizing rofecoxib are described in theliterature and in patent references. See International PatentApplication Publication No. WO 98/00416. which is incorporated herein inits entirety. Some of the key results from the lab-scale development ofrofecoxib manufacturing process A₁ are summarized below.

Some of the early laboratory trials included oxidation of4′-(Methylthio)acetophenone in the first step of rofecoxib synthesis.This approach may be abandoned in favor of performing the oxidation ofthe sulfide in the last bond forming step in order to decrease the levelof impurities in intermediates and rofecoxib.

Dimethylformamide (DMF) was used in prior manufacturing processes. Thishigh boiling-point Class 2 solvent may be replaced with acetonitrile (alower boiling-point Class 2 solvent) or DMSO (Class 3 solvent).

A safer alternative to bromine (Br₂) may be desired. Therefore,alternative reagents were explored and tetrabutylammonium bromide(TBAB₃) can be a useful alternative.

In one embodiment, the amount of TBAB₃ used is less than one equivalentrelative to the starting material, 4′-(Methylthio)acetophenone, in orderto prevent formation of a dibromacetone impurity.

In some embodiments, the step 1 product, RXB-Bromacetone, is notisolated because the intermediate may be a sensitizer and lachrymator.

The solvent system used in crystallization of RXB-Phenylacetate in step2 is important for reducing impurity levels and for increasing yield. Inone embodiment, a solvent system including isopropanol anddichloromethane provides the best results at lab scale.

The yield and purity of the cyclization reaction in step 3 is solventdependent. In one embodiment, solvents used for the reaction (DMSO) andcrystallization (DMSO and isopropanol) provide the best results forpurity and yield of 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone atlab scale. In addition, no drying step may be needed.

Although step 4 (oxidation) and step 5 (crystallization) result inhighly pure rofecoxib, in one embodiment the reaction conditions mayneed to be optimized to minimize the amount of the incomplete oxidationproduct, 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, observedduring scale up.

In one embodiment, only one crystal form was observed in crude rofecoxibof step 4 and crystallized rofecoxib of step 5 during the lab scaledevelopment.

Rofecoxib Characterization

The structure and physical properties of rofecoxib have been establishedby spectroscopic and solid-state characterization of the analyticalreference standard, lot SHD390-187. The molecular formula C₁₇H₁₄O₄S andstructure are confirmed by elemental analysis, high-resolution massspectrometry, nuclear magnetic resonance spectroscopy, 1R spectroscopy,and X-ray powder diffractometry. Solid-state characterization datasupport the anhydrous crystalline form.

In one embodiment, the results of the elemental analysis, shown in Table2, are consistent with the empirical formula for rofecoxib, C₁₇H₁₄O₄S.

TABLE 2 Elemental Analysis of Rofecoxib Amount of Element, (%) C H S OTheoretical^(a) 64.95 4.49 10.20 20.36  Experimental 64.90 4.55 10.1820.37^(b) ^(a)Theoretical values were determined on the basis of theexpected molecular formula of rofecoxib, C₁₇H₁₄O₄S. ^(b)The experimentalpercentage of oxygen was calculated as follows: 100%-% C-% H-% S.

In one embodiment, high-resolution mass spectrometry data were obtainedon rofecoxib in electrospray ionization positive mode (ESI-Pos) mode.The mass spectrum of rofecoxib in acetonitrile is shown in FIG. 4 . Theaccurate mass of the [M+H]⁺ ion is 315.0687 Daltons, which is a 0.3 ppmdifference from the theoretical mass, 315.0686 Daltons.

The Nuclear Magnetic Resonance (NMR) spectroscopy data also support thestructure of rofecoxib as shown in analysis in FIGS. 5A-D. FIG. 5A showsthe 600 MHz ¹H-NMR spectrum of rofecoxib in DMSO-d₆. FIG. 5B shows theexpanded 600 MHz ¹H-NMR spectrum of rofecoxib in DMSO-d₆. FIG. 5C showsthe 125 MHz ¹³C-NMR spectrum of rofecoxib in DMSO-d₆. FIG. 5D shows theexpanded 125 MHz ¹³C-NMR spectrum of rofecoxib in DMSO-d₆.

Below is the numbering scheme for assigning NMR resonances in rofecoxib

The proton and carbon resonances listed in Table 2 were assigned from2-dimensional (2D) correlation spectroscopy (COSY). heteronuclear singlequantum coherence (HSQC), and heteronuclear multiple bond correlation(HMBC) experiments. The 2-dimension spectra are shown in FIGS. 6A-C.FIG. 6A shows the H-H COSY NMR spectrum of rofecoxib in DMSO-d₆. FIG. 6Bshows the HSQC multiplicity edited NMR Spectrum of rofecoxib in DMSO-d₆.FIG. 6C shows the HMBC NMR spectrum of rofecoxib in DMSO-d.

In one embodiment, the infrared (1R) absorbance spectrum of solidrofecoxib was acquired using an Attenuated Total Reflectance (ATR) celland is shown in FIG. 7 . The analysis of the absorbance is provided inTable 3. The results of IR spectroscopy support the assigned structure.

TABLE 3 Proposed IR Absorbance Analysis of Rofecoxib Wavenumber (cm⁻¹)Tentative Assignment 3019, 2932 C—H stretch 1744 C═O stretch 1647, 1595C═C stretch 1446 C—H bending 1307, 1282 C—O stretch 1147 S═O stretch1035 In-plane C—H bending 775 Out-of-plane C—H bending

In one embodiment, rofecoxib is a single crystal form (Form A), which isthe only structure reported in the Cambridge Structural Database asshown in FIG. 8 . See Groom C. R., Bruno M P, Lightfoot S C. et al. TheCambridge Structural Database. Acta Cryst. 2016; B72:171-179. Theoverlay plot shown in FIG. 9 shows that the X-ray powder diffraction(XRPD) patterns of the manufactured rofecoxib reference standardcompares favorably to the XRPD pattern that was calculated from thecrystal structure reported in the Cambridge Structural Database.

In one embodiment, the analytical procedure employed herein usesreversed-phase ultra-high performance liquid chromatography withgradient elution and UV detection at 290 nm for the determination ofidentity, assay and impurities of rofecoxib drug substance. Theseparation is performed using an acid-modified, polar mobile phase on aphenyl-hexyl column. Assay is calculated using an external referencestandard. Impurities are reported as area percent. Identity is confirmedby comparison of the sample retention time to that of the referencestandard.

The physical properties of rofecoxib are provided in Table 4.

TABLE 4 Rofecoxib Specification Test Item Analytical ProcedureAcceptance Criteria Appearance Visual inspection White to off-white tolight yellow powder Identification FTIR; USP <197> Confirms to referenceIdentification UHPLC Confirms to reference Assay UHPLC 95.0-105.0 % w/won the anhydrous basis Impurities UHPLC Any Individual Impurity ≤0.50 %area Total Impurities ≤1.0 % area Water Coulometric titration Reportresult Content (KF); USP <921>, Method IC Residual GC Acetonitrile ≤410ppm Solvents Dichloromethane ≤600 ppm Dimethylsulfoxide ≤5000 ppmIsopropanol ≤5000 ppm Methanol ≤3000 ppm Polymorphic XRPD;USP <941>Conforms to reference Form (Form A) Particle size Laser diffraction,Report result USP <429> Residue USP <281> ≤0.1 % w/w on Ignition

The thermal properties of rofecoxib were evaluated using differentialscanning calorimetry (DSC). In one embodiment, rofecoxib was heated from30 to 400° C. at a ramp rate of 4° C. min⁻¹. The DSC thermogram in FIG.10A shows an endothermic event with onset temperature of 209° C., andpeak maxima at 201° C. corresponding to melting, and a broad exothermicevent with onset temperature of 371° C., and peak maxima at 385° C.,corresponding to decomposition. The absence of an endotherm attemperature below the melting temperature is consistent with theanhydrous crystal structure of rofecoxib.

Thermogravimetric analysis (TGA) was performed by heating rofecoxib fromambient to 500° C. at a ramp rate of 10° C. min⁻¹. The TGA data as shownin FIG. 10B indicate no weight loss prior to or during the melt; thisresult is consistent with the anhydrous crystal structure of rofecoxib.The weight loss at temperatures above 370° C. corresponds todecomposition of rofecoxib.

Residual DMSO in rofecoxib drug substance is quantified against anexternal standard using direct-injection gas chromatography and FIDdetection. The separation is performed using a dimethylpolysiloxanecolumn and helium carrier gas. Residual acetonitrile, dichloromethane,isopropanol, and methanol in rofecoxib drug substance are quantifiedagainst external standards using headspace gas chromatography and FIDdetection. The separation is performed using a bonded-wax column andhelium carrier gas.

Formulation

In certain aspects, the substantially pure or highly pure rofecoxib asprovided herein can be formulated as a pharmaceutically acceptable saltor solvate thereof to produce a pharmaceutical composition. In certainaspects, the substantially pure or highly pure rofecoxib or apharmaceutically acceptable salt or solvate thereof as provided hereinmay be formulated with a pharmaceutically acceptable carrier to producea pharmaceutical composition. Pharmaceutical compositions comprisingsubstantially pure or highly pure rofecoxib or a pharmaceuticallyacceptable salt or solvate thereof as provided herein may include theexcipients, and may otherwise be formulated, as described in U.S. Pat.No. 6,063,811. which is incorporated herein by reference in itsentirety, including but not limited to the formulations specified inExamples 2, 2a, 2b, and 2c of U.S. Pat. No. 6,063,811. The term“pharmaceutically-acceptable salt,” in this respect, refers to therelatively non-toxic, inorganic and organic acid salts of compounds ofthe subject matter described herein.

In some embodiments, the compounds of the subject matter describedherein may contain one or more acidic functional groups and, thus, arecapable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of compounds of the subject matterdescribed herein. These salts can likewise be prepared in situ duringthe final isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form with a suitablebase, such as the hydroxide, carbonate or bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with apharmaceutically-acceptable organic primary, secondary, or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts, andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, and the like. See, forexample, Berge et al., supra.

Formulations of the subject matter described herein include but are notlimited to those suitable for oral, nasal, inhalation, topical(including buccal and sublingual), rectal, vaginal, and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of active ingredient which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated and the particular mode of administration.The amount of active ingredient, which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Generally, out of100%, this amount will range from about 1% to about 99% of activeingredient, preferably from about 5% to about 70%, most preferably fromabout 10% to about 30%.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the subject matter describedherein with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the subject matterdescribed herein with liquid carriers, or finely divided solid carriers,or both, and then, if necessary, shaping the product.

Formulations of the subject matter described herein suitable for oraladministration may be in the form of capsules, cachets, chewable gels,pills, tablets, lozenges (using a flavored basis, usually sucrose andacacia or tragacanth), powders, granules, or as a solution or asuspension in an aqueous or non-aqueous liquid, or as an oil-in-water orwater-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles(using an inert base, such as gelatin and glycerin, or sucrose andacacia), and/or as mouthwashes and the like, each containing apredetermined amount of a compound of the subject matter describedherein as an active ingredient. A compound of the subject matterdescribed herein may also be administered as a bolus, electuary orpaste.

In solid dosage forms of the subject matter described herein for oraladministration (capsules, tablets, pills, dragees, powders, granules,and the like), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as microcrystallinecellulose, lactose, or dicalcium phosphate, and/or any of the following:fillers or extenders, such as starches, lactose. microcrystallinecellulose sucrose, glucose, mannitol, and/or silicic acid; binders, suchas, for example, carboxymethylcellulose, hydroxypropylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia;humectants, such as glycerol: disintegrating agents, such as agar-agar,calcium carbonate, croscarmellose sodium, potato or tapioca starch,alginic acid, certain silicates, sodium carbonate, and sodium starchglycolate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as, for example, cetyl alcohol, glycerol monostearate, sodiumlauryl sulfate, and polyethylene oxide-polybutylene oxide copolymer;absorbents, such as kaolin and bentonite clay; lubricants, such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxybutylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the subject matter described herein, such as dragees,capsules, pills, and granules, may optionally be scored or prepared withcoatings and shells, such as enteric coatings and other coatings wellknown in the pharmaceutical-formulating art. They may also be formulatedso as to provide slow or controlled release of the active ingredienttherein using, for example, hydroxybutylmethyl cellulose in varyingproportions, to provide the desired release profile, other polymermatrices, liposomes, and/or microspheres. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions, which can be dissolved in sterile water or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions, which can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms for oral administration of the compounds of thesubject matter disclosed herein include pharmaceutically-acceptableemulsions, microemulsions, solutions. suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isobutyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, butylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Additionally,cyclodextrins, e.g., hydroxybutyl-β-cyclodextrin, may be used tosolubilize compounds.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters. microcrystalline cellulose, aluminummetahydroxide, bentonite, agar, and tragacanth, and mixtures thereof.

Dosage forms for the topical or transdermal administration of a compoundof the subject matter disclosed herein include powders, sprays,ointments, pastes, creams, lotions, gels. solutions, patches, andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of the subject matter disclosed herein, excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of the subjectmatter disclosed herein, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the subject matter disclosed herein to thebody. Such dosage forms can be made by dissolving, or dispersing thepharmaceutical agents in the proper medium. Absorption enhancers canalso be used to increase the flux of the pharmaceutical agents of thesubject matter disclosed herein across the skin. The rate of such fluxcan be controlled, by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions, and thelike, are also contemplated as being within the scope of the subjectmatter disclosed herein.

Pharmaceutical compositions of the subject matter disclosed hereinsuitable for parenteral administration comprise one or more compounds ofthe subject matter disclosed herein in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions, or emulsions; or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, or solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection, which may be accomplished by the use of a liquid suspensionof crystalline or amorphous material having poor water solubility. Therate of absorption of the drug then depends upon its rate ofdissolution, which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. One strategy for depot injectionsincludes the use of polyethylene oxide-polypropylene oxide copolymerswherein the vehicle is fluid at mom temperature and solidifies at bodytemperature.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly (orthoesters) and poly (anhydrides). Depot-injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

When the compounds of the subject matter disclosed herein areadministered as pharmaceuticals, to subjects, they can be given per seor as a pharmaceutical composition containing, for example, 0.1% to99.5% (more preferably, 0.5% to 90%) of active ingredient in combinationwith a pharmaceutically-acceptable carrier.

The compounds and pharmaceutical compositions of the subject matterdisclosed herein can be employed in combination therapies, that is, thecompounds and pharmaceutical compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. The particular combination oftherapies (therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill also be appreciated that the therapies employed may achieve adesired effect for the same disorder (for example, the compound of thesubject matter disclosed herein may be administered concurrently withanother anticancer agents).

The compounds of the subject matter disclosed herein may be administeredintravenously, intramuscularly, intraperitoneally, subcutaneously,topically, orally, or by other acceptable means. The compounds may beused to treat arthritic conditions in mammals (e.g., humans, livestock,and domestic animals), racehorses, birds, lizards, and any otherorganism which can tolerate the compounds.

The subject matter disclosed herein also provides a pharmaceutical packor kit comprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the subject matterdisclosed herein. Optionally associated with such container(s) can be anotice in the form prescribed by a governmental agency regulating themanufacture, use. or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use, orsale for human administration.

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate, magnesium stearate, and polyethylene oxide-polybutylene oxidecopolymer, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives, andantioxidants can also be present in the pharmaceutical compositionsdescribed herein.

In one embodiment, the pharmaceutical compositions useful according tothe methods of the subject matter described herein can be formulated inany manner suitable for pharmaceutical use.

In one embodiment, the formulations of the subject matter disclosedherein can be administered in pharmaceutically-acceptable solutions,which may routinely contain pharmaceutically-acceptable concentrationsof salt, buffering agents, preservatives, compatible carriers,adjuvants, and optionally other therapeutic ingredients.

Administration

Some aspects of the subject matter disclosed herein involveadministering a pharmaceutical composition comprising an effectiveamount of an active agent to a subject to achieve a specific outcome.

For use in therapy, an effective amount of the compound can beadministered to a subject by any mode allowing the compound to be takenup by the appropriate target cells. “Administering” the pharmaceuticalcomposition of the subject matter described herein can be accomplishedby any means known to the skilled artisan. Specific routes ofadministration include, but are not limited to, oral, transdermal (e.g.,via a patch), parenteral injection (subcutaneous, intradermal,intramuscular, intravenous, intraperitoneal, intrathecal, etc.), ormucosal (intranasal, intratracheal, inhalation, intrarectal,intravaginal, etc.). An injection can be in a bolus or a continuousinfusion.

For example, the pharmaceutical compositions according to the subjectmatter disclosed herein can be administered by intravenous,intramuscular, or other parenteral means. They can also be administeredby intranasal application, inhalation, topically, orally, or asimplants; even rectal or vaginal use is possible. Suitable liquid orsolid pharmaceutical preparation forms are, for example, aqueous orsaline solutions for injection or inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols. pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops, or preparations with protracted release ofactive compounds in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of present methods for drug delivery. See Langer R (1990) Science249:1527-33. which is incorporated herein by reference in its entirety.

The pharmaceutical compositions disclosed herein can be prepared andadministered in dose units. Liquid dose units are vials or ampoules forinjection or other parenteral administration. Solid dose units aretablets, capsules, powders, and suppositories. For treatment of asubject, different doses may be necessary depending on activity of thecompound, manner of administration, purpose of the administration (i.e.,prophylactic or therapeutic), nature and severity of the disorder, ageand body weight of the subject. The administration of a given dose canbe carried out both by single administration in the form of anindividual dose unit or else several smaller dose units. Repeated andmultiple administration of doses at specific intervals of days, weeks,or months apart are also contemplated by the subject matter describedherein.

The pharmaceutical compositions described herein can be administered perse (neat) or in the form of a pharmaceutically-acceptable salt. Whenused in medicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically-acceptable salts can conveniently be used toprepare pharmaceutically-acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic. succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Compositions suitable for parenteral administration conveniently includesterile aqueous preparations, which can be isotonic with the blood ofthe recipient. Among the acceptable vehicles and solvents are water,Ringer's solution, phosphate buffered saline, dextrose 5% w/w, andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed mineral or non-mineral oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables. Carrierformulations suitable for subcutaneous. intramuscular, intraperitoneal,intravenous, etc, administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Company. Easton, Pa.

The compounds useful in the subject matter disclosed herein can bedelivered in mixtures of more than two such compounds. A mixture canfurther include one or more adjuvants in addition to the combination ofcompounds.

A variety of administration routes is available. The particular modeselected will depend, of course, upon the particular compound selected,the age and general health status of the subject, the particularcondition being treated, and the dosage required for therapeuticefficacy. The methods of the subject matter described herein, generallyspeaking, can be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofresponse without causing clinically unacceptable adverse effects.Preferred modes of administration are discussed above.

The compositions can conveniently be presented in unit dosage form andcan be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Other delivery systems can include time-release, delayed release, orsustained-release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids, or neutral fats such as mono-di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the subjectmatter described herein is contained in a form within a matrix such asthose described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152,and (b) diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

In one aspect, the pharmaceutical composition comprising rofecoxib asprovided herein can be administered in a variety of manners, includingwithout limitation orally, by subcutaneous or other injection,intravenously or parenterally. The form in which the drug will beadministered (e.g., tablet, capsule, solution, suspension, emulsion)will depend on the route by which it is administered. In one aspect, thesubject matter disclosed herein includes a pharmaceutical compositioncomprising substantially pure or highly pure rofecoxib as providedherein and a pharmaceutically acceptable carrier, wherein thepharmaceutical composition is in the form of a tablet, and wherein theamount of the rofecoxib as provided herein is 12.5 mg or 25 mg. Inanother aspect, the subject matter disclosed herein includes apharmaceutical composition comprising substantially pure or highly purerofecoxib as provided herein and a pharmaceutically acceptable carrier,wherein the pharmaceutical composition is in the form of a tablet, andwherein the amount of the rofecoxib as provided herein is about 1 mg, 2mg, 3 mg, 5 mg, 6.25 mg, 7.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg,12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg,17 mg, 17.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22.5 mg, 25 mg, 30 mg,35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, or 70 mg.

In another aspect, the subject matter disclosed herein includes apharmaceutical composition comprising substantially pure or highly purerofecoxib as provided herein and a pharmaceutically acceptable carrier,wherein the pharmaceutical composition is in the form of a tablet, andwherein the amount of the rofecoxib as provided herein is about 0.10mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, or0.70 mg/kg.

In one aspect, the pharmaceutical composition comprising rofecoxib asprovided herein may be packaged with a set of instructions warning thesubject of cardiovascular and/or gastrointestinal risks associated withadministration of the composition.

The formulations, both for human medical use and veterinary use, ofcompounds according to the subject matter described herein typicallyinclude such compounds in association with a pharmaceutically acceptablecarrier.

As used herein, the phrase “pharmaceutically-acceptable carrier”includes but is not limited to a pharmaceutically-acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, involved in carrying ortransporting the subject pharmaceutical agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable.” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: sugars, such as lactose, microcrystalline cellulose,glucose, and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose, and cellulose acetate; powdered tragacanth; malt:gelatin: talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, and soybean oil; glycols, such as butylene glycol;polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. The term “carrier” denotes anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thecomponents of the pharmaceutical compositions also are capable of beingcomingled with the compounds of the present subject matter, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficiency.

The carrier should be “acceptable” in the sense of being compatible withcompounds of the subject matter described herein and not deleterious tothe recipient. Pharmaceutically acceptable carriers, in this regard, areintended to include any and all solvents, dispersion media, coatings,absorption delaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional medium or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds (identified or designed according to the subject matterdisclosed herein and/or known in the art) also can be incorporated intothe compositions. The formulations can conveniently be presented indosage unit form and can be prepared by any of the methods well known inthe art of pharmacy. In general, some formulations are prepared bybringing the compound into association with a liquid carrier or a finelydivided solid carrier or both, and then, if necessary, shaping theproduct into the desired formulation. A pharmaceutical composition ofthe subject matter disclosed herein should be formulated to becompatible with its intended route of administration. Solutions orsuspensions can include the following components: a sterile diluent suchas water, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

A wide variety of formulations and administration methods, including,e.g., intravenous formulations and administration methods can be foundin S. K. Niazi, ed., Handbook of Pharmaceutical Formulations, Vols. 1-6[Vol. 1 Compressed Solid Products, Vol. 2 Uncompressed Drug Products.Vol. 3 Liquid Products, Vol. 4 Semi-Solid Products, Vol. 5 Over theCounter Products, and Vol. 6 Sterile Products], CRC Press. Apr. 27,2004.

Useful solutions for oral administration can be prepared by any of themethods well known in the pharmaceutical art, described, for example, inRemington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company,1990). Formulations of the subject matter described herein suitable fororal administration can be in the form of: discrete units such ascapsules, gelatin capsules, sachets, tablets, troches, or lozenges, eachcontaining a predetermined amount of the drug; a powder or granularcomposition; a solution or a suspension in an aqueous liquid ornon-aqueous liquid; or an oil-in-water emulsion or a water-in-oilemulsion. The drug can also be administered in the form of a bolus,electuary or paste, or a topical composition comprising, e.g., a creamor gel. A tablet can be made by compressing or molding the drugoptionally with one or more accessory ingredients. Compressed tabletscan be prepared by compressing, in a suitable machine, the drug in afree-flowing form such as a powder or granules, optionally mixed by abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered drug and suitable carrier moistened with an inert liquiddiluent.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients. Oral compositions preparedusing a fluid carrier for use as a mouthwash include the compound in thefluid carrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such ashydroxypropylcellulose, gum tragacanth or gelatin; an excipient such asstarch or lactose; a disintegrating agent such as croscarmellose sodiumalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF. Parsippany, N.J.) or phosphate buffered saline (PBS). Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions can be formulated in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the subject matter disclosed herein aredictated by and directly dependent on the unique characteristics of theactive compound and the therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals. Furthermore, administration can be byperiodic injections of a bolus, or can be made more continuous byintravenous, intramuscular or intraperitoneal administration from anexternal reservoir (e.g., an intravenous bag).

Topical compositions can be formulated as creams, ointments, jellies,solutions or suspensions, etc.

Methods of Use

A pharmaceutical composition comprising rofecoxib as presented hereinmay be used in the treatment or prevention of conditions or diseases insubjects, including humans.

In one aspect, the subject matter disclosed herein includesadministering a pharmaceutical composition comprising rofecoxib having afavorable impurity profile to a subject to treat or prevent a disease orcondition, including but not limited to one of the following:osteoarthritis, rheumatoid arthritis, analgesia, juvenile idiopathicarthritis, including systemic juvenile idiopathic arthritis, migraine orheadaches, juvenile rheumatoid arthritis, ankylosing spondylitis, acutepain, primary dysmenorrhea, psoriatic arthritis and fibromyalgia.

In other aspects, the disease or condition is pain-associated with acondition caused by a bleeding disorder, including migraine associatedwith von Willebrand disease. In another aspect, a subject receivingtreatment for migraine associated with von Willebrand disease expressesvon Willebrand factor at a level about 50% below normal.

In one aspect, the treatment described herein may be administered to asubject of any age. In another aspect, the subject is age 2 or older, orage 12 years or older. In another aspect, the subject is of age 12 to 75years old, inclusive.

In one aspect, the subject is screened for all or certain of the studyprotocol inclusion or exclusion criteria described below as part of thetreatment.

In another aspect, the subject is within a subject population that has areduced risk of arterial thrombosis, cardiovascular thrombotic events,or other serious cardiovascular disease or events, for example subjectswith inherited bleeding disorders or coagulopathies such as hemophiliaor von Willebrand disease, or subjects with medically-induced bleedingdisorders or coagulopathies.

In one aspect, the subject is screened for a history or current symptomsof cardiovascular disease. In one aspect, if the subject is determinedto have a history or current symptoms of cardiovascular disease, thesubject is not administered the pharmaceutical composition. In anotheraspect, if it is determined that the subject does not have a history orcurrent symptoms of cardiovascular disease, the subject is administeredthe pharmaceutical composition as further set forth herein. In yetanother aspect, the subject is screened for one or more risk factorsthat would increase the likelihood of the subject having a seriouscardiovascular thrombotic event following administration of thepharmaceutical composition as further set forth herein. In one aspect,if it is determined that the subject may be safely administered thepharmaceutical composition as further set forth herein withoutincreasing the likelihood of the subject having a serious cardiovascularthrombotic event, then the subject is administered the pharmaceuticalcomposition as further set forth herein.

In another aspect, the subject is screened for a history or currentsymptoms of gastrointestinal bleeding, ulceration, and perforation. Inone aspect, if the subject is determined to have a history or currentsymptoms of gastrointestinal bleeding, ulceration, and perforation, thesubject is not administered the pharmaceutical composition. In anotheraspect, if it is determined that the subject does not have a history orcurrent symptoms of gastrointestinal bleeding. ulceration, andperforation, the subject is administered the pharmaceutical compositionas further set forth herein.

The subject may be screened for a history or current symptoms of bothcardiovascular disease or gastrointestinal bleeding, ulceration, andperforation, in addition to any of the study protocol inclusion orexclusion criteria listed below.

A pharmaceutical composition comprising rofecoxib that is administeredfor any of the diseases or conditions described herein may besubstantially pure or highly pure, or may be essentially free of, orfree of, one or more of the impurities described herein.

In another aspect, a pharmaceutical composition comprising rofecoxib asprovided herein is administered to a subject who has mild, moderate, orsevere pain associated with a condition caused by a bleeding disorder.Pain may be measured through any clinically-validated pain assessmentmeasure. In one aspect, pain is measured through the Pain IntensityNumerical Rating Scale. In another aspect, pain associated with aspecific condition caused by a bleeding disorder, hemophilicarthropathy, is measured through the Pain Intensity Numerical RatingScale or the Patient Assessment of Arthropathy Pain (Visual AnalogScale; VAS).

In one aspect, a pharmaceutical composition comprising rofecoxib asprovided herein is administered to a subject who has pain associatedSJIA. In another aspect, a pharmaceutical composition comprisingrofecoxib as provided herein is administered to a subject who hasmigraine associated with von Willebrand disease, wherein the subjectreceiving treatment expresses von Willebrand factor at a level about 50%below normal.

In one aspect, the treatment of the subject matter disclosed hereinincludes administration of a pharmaceutical composition comprising about12.5 mg of rofecoxib as provided herein per day. In another aspect, thetreatment includes the administration of a pharmaceutical compositioncomprising about 25 mg of rofecoxib as provided herein per day. Inanother aspect, the treatment includes the administration of apharmaceutical composition comprising about 1 mg, 2 mg, 3 mg, 5 mg, 6.25mg, 7.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 20mg, 20.5 mg, 21 mg, 21.5 mg, 22.5 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg,50 mg, 55 mg, 60 mg, 65 mg, or 70 mg of rofecoxib as provided herein perday. Treatment may be administered once daily in the form of one or moretablets. In other aspects, the pharmaceutical composition comprisingrofecoxib as provided herein is administered two times or more daily.

In one aspect, a treatment regimen is provided for the safe treatment ofpain, inflammation, migraine and/or arthritis. The pain, inflammation,migraine and/or arthritis may be associated with a disease or conditioncaused by a bleeding disorder. In one aspect, the treatment subject is ahuman patient of any age. In another aspect, the patient is age 12 yearsor older.

The treatment regimen may comprise the administration of an initial (orfirst) dose of a pharmaceutical composition comprising 10 mg, 10.5 mg,11 mg, 11.5 mg, 12 mg, or 12.5 mg of rofecoxib once daily as furtherdescribed herein. The treatment regimen may further comprise evaluatingthe subject after administration of the initial dose to determine if theinitial dose was fully, partially, or not effective at treating thepain, inflammation, migraine and/or arthritis. In another aspect, thetreatment regimen may comprise determining if the subject could benefitfrom the administration of a higher dose of rofecoxib. The evaluationand determination steps may take place after a single administration ofthe initial dose, or after multiple administrations of the initial dose(e.g. two days, three days, one week, two weeks, or longer after thefirst administration of the initial dose), and may be performed by aphysician, physician's assistant, nurse, or other health care provider.In one aspect, the evaluation and determination steps may be based onsubject-reported outcomes, and may include an assessment of the benefitof a higher dose of rofecoxib compared to any potential safety risksassociated with that higher dose. For example, if a subject experiencesa clinically meaningful decrease in pain after administration of theinitial dose, it may be determined that the subject should continue onthe initial dose through the duration of the bleeding episode thatcaused the pain.

The treatment regimen may further comprise the administration of asubsequent (or second) dose of a pharmaceutical composition comprising17.5 mg, 20 mg, or 25 mg of rofecoxib once daily if it was determinedthat the initial dose was not effective or only partially effective attreating the pain, inflammation, migraine and/or arthritis, or if it isdetermined that the subject could benefit from a higher daily dose ofrofecoxib to treat the pain, inflammation, migraine and/or arthritis(e.g. that a higher dose could achieve a greater reduction in pain inthe subject). In one aspect, the subsequent dose is administered if itis determined that the initial dose did not achieve a clinicallymeaningful reduction in pain, inflammation, migraine and/or arthritis inthe subject. In another aspect, the subsequent dose is administered ifit is determined that the subsequent dose may increase the effectivenessof the treatment without increasing the risk of adverse events or otherside effects. In another aspect, a higher dose is not administered if itis determined that the initial dose was effective at treating the pain,inflammation, migraine and/or arthritis. In another aspect, a higherdose is not administered if it is determined that the higher dose wouldincrease the risk of adverse events or other side effects in thesubject. In another aspect, a higher dose is not administered if it isdetermined that the risk of administering the higher dose (e.g. in termsof adverse events or side effects) outweigh the benefits (e.g. in termsof effectiveness of treating the pain, inflammation, migraine and/orarthritis). In another aspect, the step of not administering a higherdose includes instructing the subject not to take a higher dose of thepharmaceutical composition (e.g. not to take 12.5 mg of thepharmaceutical composition more than once daily).

In one aspect, the treatment includes the administration ofpharmaceutical composition comprising about 0.10 mg/kg, 0.15 mg/kg, 0.20mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, or 0.70 mg/kg.

In one aspect, an effective amount of rofecoxib as provided herein fortreating pain associated with a disease or condition caused by ableeding disorder is about 12.5 mg once daily. and, in another aspects,results fewer side effects or in a reduction of pain equal to or betterthan the use of a pharmaceutical composition comprising about 25 mgrofecoxib that is not substantially pure or highly pure, or essentiallyfree of, or free of, one or more of the impurities described herein thatwas present in previously available rofecoxib bulk drug product. In oneaspect, an effective amount of rofecoxib as provided herein for treatingpain associated with a disease or condition caused by a bleedingdisorder is about 17.5 mg once daily, and, in another aspects, resultsfewer side effects or in a reduction of pain equal to or better than theuse of a pharmaceutical composition comprising about 25 mg rofecoxibthat is not substantially pure or highly pure, or essentially free of,or free of, one or more of the impurities described herein that waspresent in previously available rofecoxib bulk drug product. In oneaspect, an effective amount of rofecoxib as provided herein for treatingpain associated with a disease or condition caused by a bleedingdisorder is about 20 mg once daily, and, in another aspects, resultsfewer side effects or in a reduction of pain equal to or better than theuse of a pharmaceutical composition comprising about 25 mg rofecoxibthat is not substantially pure or highly pure, or essentially free of,or free of, one or more of the impurities described herein that waspresent in previously available rofecoxib bulk drug product. In oneaspect, an effective amount of rofecoxib as provided herein for treatingpain associated with a disease or condition caused by a bleedingdisorder is about 1 mg, 2 mg, 3 mg, 5 mg, 6.25 mg, 7.5 mg, 10 mg, 10.5mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5mg, 22.5 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65mg, or 70 mg once daily. As a result, a subject may not need to beadministered the higher quantity of active ingredient in order toexperience a reduction in pain.

In one aspect, an effective amount rofecoxib as provided herein fortreating pain associated with a disease or condition caused by ableeding disorder, pain associated with juvenile idiopathic arthritis,including systemic juvenile idiopathic arthritis, or migraine associatedwith von Willebrand disease is about 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg,0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50 mg/kg,0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, or 0.70 mg/kg.

In one aspect, the treatment described herein is effective at treatingmild, moderate, or severe pain in a subject without theco-administration of another pain medication or analgesic.

In another aspect, the treatment described herein results in the subjectdecreasing or discontinuing the use of another pain medication oranalgesic, including rescue medications, during the course of thetreatment when compared to before the initiation of the treatment. Inyet another aspect, the treatment results in the subject decreasing ordiscontinuing the use of acetaminophen and/or opioid medications duringthe treatment during the course of the treatment when compared to beforethe initiation of the treatment.

In one aspect, a pharmaceutical composition comprising rofecoxib asprovided herein is co-administered with factor replacement therapy to asubject having a bleeding disorder. In another aspect, the treatmentdescribed herein is administered to a subject having a bleeding disorderwho is being administered or is taking factor replacement therapyprophylactically. In one aspect, the pharmaceutical compositioncomprising 12.5 mg, 17.5 mg, 20 mg or 25 mg rofecoxib as provided hereinis administered once daily to a subject who is also being administeredor is taking factor replacement therapy prophylactically.

In one aspect, a pharmaceutical composition comprising rofecoxib asprovided herein is administered daily and does not increase risk ofcardiovascular diseases and/or gastrointestinal bleeding, ulceration, orperforation during the course of the treatment, as determined at 2weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 52 weeks, and/or two ormore years. In another aspect, the rofecoxib as provided herein may beadministered during the course of the treatment without the use orco-administration of a gastro-protective agent including but not limitedto an antacid therapy, an H2 antagonist, a proton pump inhibitor, ormisoprostol.

In another aspect, a pharmaceutical composition comprising rofecoxib asprovided herein is administered only on an as-needed basis, for examplewhen a subject experiences a pain “flare” described as an increase inpain rating of >1 or a pain rating of ≥4 to ≥59 based on the PainIntensity Numerical Rating Scale. In yet another aspect, thepharmaceutical composition comprising rofecoxib as further set forthherein is not administered as a maintenance therapy, prophylactically,or for long term use (e.g., >1 year). In one aspect, a pharmaceuticalcomposition comprising rofecoxib as provided herein is administered onlyon an as-needed basis and for short term use, for example, less than oneweek, two weeks, three weeks, or four weeks, or until the pain,migraine, arthritis, inflammation, or other conditions or symptomssubside or resolve, for example, until there is a clinically significantimprovement in pain rating based on the based on the Pain IntensityNumerical Rating Scale.

In one aspect, the subject uses or is co-administered agastro-protective agent during the course of treatment with apharmaceutical composition comprising rofecoxib as provided herein,which prevents or treats gastrointestinal bleeding, ulceration, andperforation in the subject. In another aspect, the subject uses or isco-administered an antiviral therapy such as fameiclovir or penciclovirduring the course of treatment with a pharmaceutical compositioncomprising rofecoxib as provided herein to treat or preventfibromyalgia.

In one aspect, the treatment described herein achieves a reduction in atleast 1 from baseline in the Pain Intensity Numerical Rating Scale. Inanother aspect, the treatment described herein achieves a reduction inat least 2, 3, 4, or 5 from baseline in the Pain Intensity NumericalRating Scale.

In one aspect, the reduction in the Pain Intensity Numerical RatingScale is achieved within 1, 2, 3, 4, 5, or 6 days, or 1 week, or 2 weeksof first administering the pharmaceutical composition.

In one aspect, the treatment of a disease or condition by theadministering of a pharmaceutical composition comprising substantiallypure or highly pure rofecoxib does not result in one or more of thefollowing adverse events: upper respiratory infection, headache, nausea,vomiting, and cough, or one or more of the following serious adverseevents: hemorrhage and hypotension. In one aspect, the treatment of adisease or condition caused by a bleeding disorder by the administeringof a pharmaceutical composition comprising substantially pure or highlypure mfecoxib does not result in an increased number of joint bleedingevents. In another aspect, the treatment of a disease or conditioncaused by a bleeding disorder by the administering of a pharmaceuticalcomposition comprising substantially pure or highly pure rofecoxib doesnot increase the risk of joint bleeding events. In one aspect, thetreatment of a disease or condition caused by a bleeding disorder by theadministering of a pharmaceutical composition comprising substantiallypure or highly pure rofecoxib does not result in an increase in theamount of factor use in the subject. In another aspect, the treatment ofa disease or condition by the administering of a pharmaceuticalcomposition comprising substantially pure or highly pure rofecoxib doesnot result in an increased risk of side effects (including but notlimited to hemorrhaging, hypotension or serious cardiovascularthrombotic events) compared to the previously marketed “VIOXX” productwhen used in that disease or condition. In another aspect, apharmaceutical composition comprising substantially pure or highly purerofecoxib as provided herein results in greater efficacy in a disease orcondition compared to the previously marketed “VIOXX” product when usedin that disease or condition (as measured by a clinically-validatedmeasure, such as the Pain Intensity Numerical Rating Scale).

In another aspect, the treatment of a disease or condition by theadministering of a pharmaceutical composition comprising highly purerofecoxib that is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione does not result inone or more of the following adverse events: upper respiratoryinfection, headache, nausea, vomiting, and cough, or one or more of thefollowing serious adverse events: hemorrhage and hypotension. In anotheraspect, a pharmaceutical composition comprising highly pure rofecoxibthat is essentially free of, or free of,4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and/or4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione as provided hereinresults in greater efficacy in a disease or condition, or reduced sideeffects (e.g. hemorrhaging, hypotension or serious cardiovascularthrombotic events) compared to the previously marketed “VIOXX” productwhen used in that disease or condition (as measured by aclinically-validated measure, such as the Pain Intensity NumericalRating Scale). The purity of the resulting rofecoxib described as hereinis determined as a percent area basis, typically as quantified byanalytical chromatography, such as using HPLC. UHPLC, UPLC or otheranalytical means in the art.

EXAMPLES Example 1—Protocol for Treating Hemophilic Arthropathy (HA)

Study Objective: Evaluate the efficacy of a pharmaceutical compositioncomprising rofecoxib as provided herein (the study drug) versus placeboin subjects with hemophilic arthropathy.

Secondary objectives include:

-   -   To evaluate the effect on sleep interference due to pain,        subject global impression, and quality of life in subjects        treated with rofecoxib as provided herein versus placebo.    -   To evaluate the safety and tolerability of rofecoxib as provided        herein in subjects with hemophilic arthropathy.    -   To evaluate the long-term safety and efficacy of 2 dose regimens        of rofecoxib as provided herein in subjects with hemophilic        arthropathy.        Study Endpoints:

Primary endpoint: Change from baseline to Week 12 in the weekly averageof the daily pain score as measured using a Pain Intensity NumericalRating Scale [PI-NRS], an 11-point numerical scale where 0=no pain and10=pain as bad as can be imagined.

Secondary endpoints: Change from baseline to Month 15 in the weeklyaverage of the daily pain score as measured using a Pain IntensityNumerical Rating Scale [PI-NRS]

Secondary endpoints at Week 12 and Month 15:

-   -   30% and 50% responders on the weekly average of the daily pain        score    -   Change from baseline in the weekly average of the daily sleep        interference score.    -   Percentage of subjects who are much or very much improved using        the Patient Global Impression of Change (PGIC)    -   Eurogol (EQ-5D-5L)    -   SF-36

Safety endpoints (during the double blind and open-label phaseseparately):

-   -   Incidence of thrombotic events    -   Incidence of GI bleeding events    -   Incidence of any bleeding event    -   Adverse Events. Laboratory Safety Tests (hematology,        coagulation, clinical chemistry), blood pressure, pulse rate,        ECG, C-SSRS    -   Factor use    -   Percentage of subjects who discontinue due to adverse events.

Other endpoints:

-   -   Percentage of subjects who discontinue due to lack of efficacy    -   Average acetaminophen usage per day and percentage of subjects        using rescue medication.

Study Design: Multicenter, double-blind, randomized, placebo-controlled.parallel-group study to evaluate the efficacy and safety of rofecoxib asprovided herein in subjects with hemophilic arthropathy. Eligiblesubjects must have a diagnosis of hemophilia A. B (factor VIII or factorIX deficiency with or without inhibitor) or von Willebrand disease (VonWillebrand factor level of ≤30 IU per dL). a history of joint bleeding,chronic symptomatic pain in one or more joints on 20 of the 30 daysprior to screening, and a diagnosis of hemophilic arthropathy at least6-months prior to screening, with the primary source of pain ordisability in the hip, knee, ankle or elbow.

At screening, the pain over the last week will be assessed using the11-point PI-NRS (0-10), and pain intensity needs to beat least 3.Following Screening, subjects who meet eligibility criteria will need todiscontinue ongoing NSAID and Cox-2 inhibitor medications at least 7days prior to randomization. Subjects who are using weak or low doseopiates or other non-NSAID analgesics are allowed to continue those atstable dose during the study.

Subjects will daily record their average pain intensity over the last 24hours in an electronic diary using the 11-point PI NRS for the durationof the study treatment period. Rescue medication can be used(paracetamol/acetaminophen) if needed up to 3 g/day. If rescuemedication is used for more than 2 consecutive days, the maximum dosageis 2.5 g. Subjects who need rescue medication for more than 7consecutive days at a dose of 2.5 g/day need to discontinue study drug.The dosage of any rescue medication must be recorded in the electronicdiary.

The weekly average of the PI-NRS collected on the 7 days prior torandomization will be defined as the baseline. Subjects with a flair ofpain intensity indicated by an increase in the weekly mean of the dailypain score of at least 1.5 points and having at least moderate painintensity (weekly average baseline arthritic pain score: ≥4 and ≤9) willbe eligible to enter the double-blind treatment period.

On Day 1, eligible subjects will be randomized in a 1:1:1 ratio toreceive rofecoxib as provided herein at 25 mg QD, rofecoxib as providedherein 12.5 mg QD, or matching placebo. Double-blind treatment willcontinue for 12 weeks. Following the initial double blind treatmentperiod, subjects will be re-randomized in a 1:1 ratio to receiverofecoxib as provided herein at 25 mg QD or 12.5 mg QD for an additional12-months. Subjects will attend a Follow Up Visit approximately 1 weekafter the last dose of study treatment, and will receive a follow upphone call 4 weeks after the last dose.

Study Population:

Inclusion Criteria

To be eligible to participate in the study, and to receive the studydrug, candidates must meet the following eligibility criteria:

-   -   1. Ability of the subject to understand the purpose and risks of        the study and provide signed and dated informed consent and        authorization to use confidential health information in        accordance with national and local subject privacy regulations.    -   2. Aged 12 to 75 years, inclusive, at the time of informed        consent.    -   3. All women of childbearing potential and all men must practice        effective contraception during the study and for 5 weeks for        women and 14 weeks for men, after their last dose of study        treatment.    -   4. Diagnosis of hemophilia A. B (factor VIII or IX deficiency        with or without inhibitor) or von Willebrand disease (Von        Willebrand factor level of ≤30 IU per dL)    -   5. History of joint bleeding    -   6. Diagnosis of hemophilic arthropathy for at least 6-months        prior to Screening with history of joint bleeding and the        primary source of pain or disability in the hip, knee. ankle or        elbow.    -   7. Chronic symptomatic pain in one or more joints on 20 of the        30 days prior to screening.    -   8. Has an intensity of 23 and 59 on the Numerical Rating Scale        based on a paper-based question at Screening that asks for the        average pain intensity of arthritic pain due to hemophilic        arthropathy over the last week.    -   9. If on analgesic medication to manage pain due to hemophilic        arthropathy, subject must have had stable analgesic medication        for a minimum of 30 days prior to Screening.    -   10. Has a flair of pain intensity indicated by an increase in        the weekly mean of the daily pain score of at least 1.5 points    -   11. Has a baseline weekly average of the daily pain score ≥4 and        ≤9 on the electronic diary PI-NRS; baseline is defined as the 7        days prior to randomization (Day 1).        Exclusion Criteria

Candidates will be excluded from study entry, and receiving the studydrug, if any of the following exclusion criteria exist:

Medical History

-   -   1. Is pregnant or lactating (female subjects only).    -   2. Known hypersensitivity to rofecoxib or any other component of        the formulation    -   3. History of asthma, urticaria, or allergic-type reactions        after taking aspirin or other NSAIDs    -   4. Has a history of advanced renal disease.    -   5. Has a history of any liver disease within the last 6-months,        with the exception of known Gilbert's disease.    -   6. Has a history of alcohol or substance abuse.    -   7. Currently has uncontrolled or poorly controlled hypertension.    -   8. Has a history of major cardiac ischemic symptoms, events or        interventions such as angina pectoris, myocardial infarction,        acute coronary syndrome, decompensated congestive heart failure,        coronary stent or bypass.    -   9. Has a history of cerebrovascular ischemic events (TIA or        stroke). Subjects with a history of intra- or extracerebral        hemorrhage may be eligible if their condition is stable.    -   10. Has a history of major vascular ischemic symptoms such as        claudicatio intermittens, or vascular bypass or replacement        surgery.    -   11. Has a history or presence of significant cardiovascular,        gastrointestinal, or renal disease, or other condition known to        interfere with the absorption, distribution, metabolism, or        excretion of drugs.    -   12. Has a history or presence of any clinically significant        abnormality in vital signs, ECG. or laboratory tests or has any        medical or psychiatric condition that, in the opinion of the        Investigator, may interfere with the study procedures or        compromise subject safety.    -   13. History of a major upper GI event (upper GI perforation,        obstruction or major upper GI bleeding) within 6-months prior to        screening    -   14. Has had an episode of major depression within 6-months        before Screening.    -   15. Has a history of suicide attempt within 6-months before        Screening.

Vitals and Laboratory Procedures

-   -   1. Has BP≥2160 mmHg systolic and/or 2100 mmHg diastolic at        Screening after repeated measurements.    -   2. Has a QT interval corrected using Fridericia's formula        (QTcF)≥2450 msec (males) or ≥2470 msec (females) [average of 3        measurements at least 5 minutes apart and done within 15        minutes] at Screening.    -   3. Has a positive pregnancy test at Screening (women of        childbearing potential only).    -   4. Estimated creatine clearance (using Cockroft-Gault        equation)<30 ml/min    -   5. Has AST or ALT≥2× the upper limit of normal (ULN) or has        alkaline phosphatase or bilirubin 1.5×ULN at Screening.    -   6. History or positive test result at Screening for human        immunodeficiency virus (HIV).    -   7. History or positive test result at Screening for hepatitis C        virus (HCV) antibody or hepatitis B virus (defined as positive        for hepatitis B surface antigen [HBsAg] or hepatitis B core        antibody [HBcAb]).    -   8. Has a positive drug screen for drugs of abuse at Screening        (amphetamine, barbiturates, benzodiazepines, cocaine, opiates,        tetrahydrocannabinol) except if explained by use of allowed        prescription medicines).        Other Screening Assessments

Has a positive response on Item 4 or 5 on the C-SSRS at Screening.

General

-   -   1. Is mentally or legally incapacitated.    -   2. Is unable to comply with the restrictions related to        prohibited concomitant therapy restrictions.    -   3. Previous registration in this study or previous studies with        rofccoxib.    -   4. Has participated in an interventional study and received        study treatment within 3-months before Screening.    -   5. Has donated blood or blood products within a 30-day period        prior to Screening.    -   6. Inability to comply with study requirements.    -   7. Other unspecified reasons that, in the opinion of the        Investigator, make the subject unsuitable for enrollment.

Medications

-   -   1. Concomitant use rifampin    -   2. Failed to discontinue concomitant NSAID or Cox-2 pain        medications 7 days prior to randomization    -   3. Has used rescue medication at a dose of 2.5 g        acetaminophen/paracetamol for more than 5 days consecutively        prior to randomization.

Baseline Pain Scores

-   -   1. Has missed more than 2 of 7 daily pain score entries during        the last 7 days of treatment prior to randomization.    -   2. Has a daily pain score of 52 on 1 or more days during the        last 7 days of treatment prior to randomization.    -   3. Has a difference between the lowest and highest daily pain        score of >4 during the last 7 days of treatment prior to        randomization.

Treatment Groups:

Subjects will be randomized to receive double-blind study drug in a1:1:1 ratio:

-   -   Rofecoxib as presented herein (25 mg QD orally)    -   Rofecoxib as presented herein (12.5 mg QD orally)    -   Placebo (matching tablets QD orally)        Concomitant Medications:

Allowed medications

-   -   PPI therapy will be provided to all study participants for        gastric protection    -   Stable low opioids or other non-NSAID pain medications

Prohibited Medications

-   -   NSAIDs or Cox-2 inhibitors

Visit Schedule: There will be 7 visits: screening visit up to 28 daysprior to start of dosing: Randomization (Day 1); Double-Blind Treatmentvisits (Weeks 4, 8 and 12); Open-Label Treatment Visits (months 4, 6, 9,12) and a Follow Up visit 7 to 10 days after the last dose. In addition,there will be a follow up phone call 28 days after the last dose.

Discontinuation of Treatment: A subject must permanently discontinuestudy treatment for any of the following reasons:

-   -   The subject becomes pregnant.    -   The subject withdraws consent to continue study treatment.    -   The subject experiences a medical emergency that necessitates        permanent discontinuation of study treatment.    -   The subject experiences a medical emergency that necessitates        unblinding of the subject's treatment assignment.    -   The subject is unwilling or unable to comply with the protocol.    -   The subject meets individual liver chemistry, vital signs or        ECG. C-SSRS or adverse event discontinuation criteria    -   At the discretion of the Investigator for medical reasons.        Efficacy Assessments:

Eleven-point PI-NRS for average daily pain (assessed daily in theevening) [PI NRS scores will be collected in the electronic diary.]

-   -   Eleven-point S-NRS score for daily sleep interference (DSIS,        assessed daily in the morning) [S NRS scores will be collected        in the electronic diary.]    -   PGIC    -   EQ-5D-5L    -   SF-36

Safety Assessments:

-   -   Incidence of thrombotic events    -   Incidence of GI bleeding events    -   Adverse Events    -   Laboratory Safety Tests (hematology, coagulation clinical        chemistry)    -   Blood Pressure    -   Pulse Rate    -   ECG    -   C-SSRS

Example 2—Batch Analysis

Batch analysis of two rofecoxib batches, batch SHD390-187 and batch16P3140F851. is summarized in Table 5. If an individual impurity is≥0.05% area, the result is reported to the nearest 0.01%. If anindividual impurity is ≤0.02% (LOD), the result is reported as “notdetected” (ND). If an individual impurity is <0.05% area but ≤0.02%area, the result is recorded as <0.05% area.

TABLE 5 Batch Analysis Data for Rofecoxib Drug Substance Lot No.Identifi- Identifi- Assay % w/w (manufactur- cation cation on anhydrousing process) Appearance by HPLC by FTIR basis SHD390-187 White NA NA NA(A₁) powder 16P3140F851 Off-white Conforms to Conforms to 99.8 (A₁)powder Reference Reference Impurities by HPLC Area % Lot No.(manufactur- Total RXB- RXB- RRT RRT ing process) Impurities FuranoneSulfoxide ~0.5 ~0.66 SHD390-187 0.2 ND 0.12 ND 0.11 (A₁) 16P3140F851 0.10.08 ND <0.05 ND (A₁) Residual Solvents by GC ppm Lot No. (manufacturAcetoni- Dichloro- Dimethyl- Isopro- Meth- ing process) trile methanesulfoxide panol anol SHD390-187 ND ND 506 14 ND (A₁) 16P3140F851 ND ND484 ND ND (A₁) Water Lot No. Content Residue on Particle Size Analysisby (manufactur- Karl Fischer Polymorphic Ignition Laser Diffraction, μming process) % w/w Form XRPD % w/w d₁₀ d₅₀ d₉₀ SHD390-187 0.0 Form A 0.0NT NT NT (A₁) 16P3140F851 0.1 Form A 0.0 0.859 3.83 11.1 (A₁)

Example 3—Stability Data for Rofecoxib

Stability data for rofecoxib drug substance (Lot 16P3140F851) areprovided in Tables 6A-B. If an individual impurity is ≥0.05% area, theresult is reported to the nearest 0.01%. If an individual impurity is<0.02% (LOD), the result is reported as “not detected” (ND). If anindividual impurity is <0.05% area but ≥0.02% area, the result isrecorded as <0.05% area.

TABLE 6A Stability of Rofecoxib Drug Substance (Lot 16P3140F851) at 40°C./75% RH Lot: 16P3140F851 Manufactured At: PCAS Batch Scale: 17.74 kgContainer/ Double HDPE Closure: bags in HDPE drum Condition: 40° C./Manufacture 3 Jul. 2018 Stability Initiation 75% RH Date: Date: 24 Aug.2018 Test Initial 1 Month 3 Month 6 Month Appearance Off-white Off-whiteWhite powder powder powder Assay by HPLC (w/w) 99.8 99.6 100.5Impurities by HPLC (% area) RXB-furanone 0.08 0.08 0.08 RRT ~ 0.5 <0.05<0.05 <0.05 Total Impurities 0.11 0.11 0.12 Water Content by KarlFischer 0.12 0.07 0.09 (%) Polymorphic form by XRPD Form A NT NT

TABLE 6B Stability of Rofecoxib Drug Substance (Lot 16P3140F851) at 25°C./60% RH Lot: 16P3140F851 Manufactured At: PCAS Batch Scale: 17.74 kgContain/ Double HDPE Closure: bags in HDPE drum Condition: 25° C./Manufacture 3 Jul. 2018 Stability Initiation Date: 60% RH Date: 24 Aug.2018 Test Initial 3 Month 6 Month 9 Month 12 Month Appearance Off-whiteWhite powder powder Assay by HPLC (% w/w) 99.8 100.4 Impurities by HPLC(% area) RXB-furanone 0.08 0.08 RRT ~ 0.5 <0.05 <0.05 Total Impurities0.11 0.11 Water Content by Karl Fischer 0.12 0.08 (%) Polymorphic formby XRPD Form A NT

Example 4—Oxidation Reaction

TABLE 7 Effect of solvents on the kinetics. VLA P075-176 VLA P075-178CHG P059-084 Materials RXB-Furanone 3.00 g 3.00 g 3.00 g H₂O₂ 35% 2.5eq. 2.5 eq. 2.5 eq. Na₂WO₄•2H₂O 1 mol % 1 mol % 1 mol % ACN 6 V 6 V 7 VIPA 36 % wt. None 36 % wt. H₂O 0.25 V 0.25 V 0.25 V Process H₂O₂addition time 2 h 20 2 h 00 2 h 00 RXB-201 seeding None 1 seed, end of 2seeds during H₂O₂ addition H₂O₂addition Aspect Comments Slightly cloudedWhite suspension White suspension during whole at the end of after firstprocess. H₂O₂ addition. seeding. Addition 1 V ACN after IPC2:whitesuspension UHPLC IPC 1 (End of 97.23/2.50 99.20/0.53 Not performed (%area) H₂O₂ addition) RXB-201/ IPC 2 (time) 99.72/n.d. 99.68/0.0299.40/n.d. Sulfoxide (1 h) (30 min) (45 min) Work-up Not isolated Notisolated 2 V water Isolated RXB-201 Not isolated Not isolated 99.85/0.02

Effect of solvents (ACN. IPA, water) on oxidation reaction completionand the effect of solvents on the kinetic are studied in several trialsas shown in Table 7. The use of 0.25V deionized water allowed to startthe reaction from the beginning of hydrogen peroxide addition. The firsttrial (VLA P075-176) was conducted using the standard process (at 65°C.), with addition of 0.25V pure water. Addition of hydrogen peroxidewas achieved in 2h 20 min. A slightly clouded reaction medium wasobtained, with a very low amount of solid in suspension. At the end ofH₂O₂ addition, 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone wasnever detected, and only 2.5%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone for this trial.After an additional hour of stirring at 65° C.,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone was not detected inUHPLC.

This trial resulted in very fast conversion of species into rofecoxib.At the end of reaction, 1V ACN was added to the reaction to try to get afull solubilization of the reaction medium. In fact, the reaction mediumturned immediately heterogeneous, as white suspension. This indicates anoversaturation of the reaction medium. Rofecoxib crystallized out withthe perturbation generated by the addition of acetonitrile.

The effect of isopropanol content was then studied in the second trial(VLA P075-178). The reaction was performed without IPA. which shouldhave a negative effect of rofecoxib species solubility. The reactionmedium was already in suspension at the end of hydrogen peroxideaddition, before seeding with rofecoxib. A co-crystallization of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(51H)-furanone and rofecoxibcould lead to a low conversion of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, with long time toreach IPC specification.

Finally, a better conversion was observed at the end of H₂O₂ additioncompared to VLA P075-176. Moreover, the H₂O₂ addition was performedfaster than the first trial (2h instead of 2h 20 min). The absence ofIPA reveals faster oxidation conditions than with 36% IPA. This could beattributed to a slightly higher concentration of reaction medium. Thecrystallization of the reaction medium seems to have no effect on the4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone oxidation kinetics.

The trial CHG P059-084 was launched in 7V ACN with usual amount of IPA.This higher dilution revealed to have no effect on reaction mediumaspect at the beginning of oxidation. Two seedings were performed duringH₂O₂ addition to get the white suspension while addition. Reactionmedium aspect was similar to VLA P075-178. 45 minutes after hydrogenperoxide addition completion,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone was not detected.After addition of 2V water as quench, cooling to 0° C., filtration andcake washings, the solid obtained contained 0.02%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. Therefore,reaction medium could be both slightly clouded or a heterogeneous whitesuspension, the IPC specification was achieved after only 1 h of heatingafter hydrogen peroxide addition completion. The oversaturation of thereaction medium is with 6V ACN seems to not be an issue for conversionof rofecoxib species into rofecoxib.

Example 5—Process Optimization—Oxidation of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone

Process optimization has led to the following oxidation process. Thedemo batch was performed on 24 g4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone in jacketed reactorequipped with mechanical stirring (CHG P059-092). The process wasperformed as shown in-FIG. 24 .

Example 6—Optimization of Rofecoxib (RXB-201) Recrystallization

Solubility and nucleation of rofecoxib was measured at four differentconcentrations: 3 V, 4 V, 5 V, and 6 V. As shown in FIG. 11 thesolubilization curve is reported in blue, and nucleation in red. Theprocess used for solubilization of rofecoxib at the beginning isreported as the purple spot (5.5V at 40° C.). In one embodiment, theproductivity can be improved by increasing the concentration to 4.5V,with heating at 50° C. (green spot). These conditions allow a goodsolubilization of rofecoxib, with a large safety margin compared to thenucleation temperature (at least 30° C.). This margin is required toavoid spontaneous crystallization during clarifying filtration. Anextra-amount of DMSO (+0.5 V) can be used for rinsing of the filtrationsystem.

Trial VLA P075-180 was conducted with rofecoxib containing a high amountof ashes (1.5%). This led to high level of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one impurity.With higher DMSO rate (6V), and lower temperature (50° C.), trial VLAP075-184 produced rofecoxib in good yield, with satisfying purge of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one impurity.These good results were obtained even with high ashes rate as trials 180and 184 were conducted on the same starting material. The trial CHGP059-090 was conducted using the process described in therecrystallyzation flowsheet below. The product was obtained in goodyield, with the slightly lower purge of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. Formation of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one was avoideddue to the clarifying filtration used.

The trials are presented in Tables 8A-B below:

TABLE 8A Study trials Spiking Solvents Sulfoxide Temperature ReferenceScale DMSO Water (% a/a) Initial Final VLA P075- 10.1 g 5.5 V 5 V 0.2360° C. 20° C. 180 VLA P075- 10.1 g 6 V 5.5 V 0.22 50° C. 0° C. 184 CHGP059- 10.0 g 5 V 5.5 V 0.47 50° C. 0° C. 090 Demo batch 5 V 5.5 V 50° C.20° C.

TABLE 8B Study trials (cont.) UHPLC results (% a/a) Purge factor (UHPLC)Yield Fura- Sulf- Hy- RXB- Fura- Hy- Sulf- Reference (%) none oxidedroxy 201 none droxy oxide VLA P075- 89 n.d. 0.10 0.47 99.43 n.a. 5.62.3 180 VLA P075- 92 n.d. 0.11 0.08 99.81 n.a. 6 2.0 184 CHG P059- 92n.d. 0.28 0.04 99.37 n.a. 10 1.7 090 Demo batch To be performed

The recrystallization flowsheet is shown in FIG. 25 .

Example 7—Further Optimization of Rofecoxib (RXB-201) RecrystallizationContext of Study

FIG. 26 shows a process flowsheet for4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation.

Optimization was conducted with respect to the following factors:

-   -   The purge factor of        4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is low. The        recrystallization process allows to half the amount of        4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone (purge        factor=2). This poor efficiency of impurity removal leads to the        need of a low specification at oxidation IPC        (4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone <0.20%        area) to get an API in specifications.    -   High volumes of solvents are used. 7 volumes of DMSO and 7.5        volumes of purified water are needed to perform the        recrystallization. The 14.5 volumes in total could be an issue        for productivity during upscale.

The first part of the optimization work has been focused on solventsscreening to find the best solvents for solubilization andcrystallization of rofecoxib (RXB-201). The purge factor of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone was then studiedwith various solvents. The formation of another impurity,4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one, was alsostudied. Finally, the optimization work was concentrated on improvementof recrystallization productivity.

Solvents Screening Assisted by Dynochem Software

The tool called “Early phase solvent selection: solubility prediction”was used. This tool allows to predict the solubility of material in alarge scope of solvents, knowing the solubility in some of them. Thesolubility can be also predicted in function of temperature, and forsolvent combinations. The solubility prediction is based on similarstructures moieties between solvents. Therefore, the measurement ofsolubility in 16 different solvents allowed for the estimate ofsolubility in up to 106 solvents. Table 9 below reports the solubilitymeasurements of rofecoxib (RXB-201) performed in 14 solvents andpredicted solubilities of anisole and isopropanol. Solubilities wereobtained by UHPLC assay. Among these 106 solvents, 27 seemed interestingas solvent or antisolvent. DMF. NMP and DMSO are the solvents in whichrofecoxib (RXB-201) is the most soluble (>100 g/L at 30° C.).Nevertheless, ICH Q3C, DMF and NMP were less suitable than DMSO whichremains the best option. Thus, no changes were implemented regarding thesolvent.

TABLE 9 Solubility of rofecoxib (RXB-201) in various solventsTemperature Solubility Solvent (° C.) (g/L) Data n-heptane 30 <0.1Measured Anisole 30 22 Measured Toluene 30 1.2 Measured Methanol 30 1.8Measured Ethanol 30 <0.1 Dynochem n-butanol 30 0.2 Measured IPA 20 0.4Dynochem Water 30 <0.1 Measured Acetone 30 28 Measured Ethyl acetate 304.7 Measured Acetonitrile 30 41 Measured Acetic acid 30 5 MeasuredDichloromethane 30 45 Measured Chlorobenzene 30 2.3 Measured DMF 30 133Measured NMP 30 140 Measured DMSO 20 93 Measured 30 107 Measured 40 133Measured 50 181 Measured 60 181 Measured

Regarding the antisolvent, rofecoxib (RXB-201) is very poorly soluble inwater (insoluble regarding US pharmacopeia). This implies rapidcrystallization occurs upon addition of water an antisolvent. It may bethe origin of the bad impurity purge. Among the solvents studied forsolubility measurements, and similarly to previous stages. IPA seems tobe a good candidate. Indeed, rofecoxib (RXB-201) is slightly moresoluble in IPA than in water, which can lead to a smoothercrystallization.

Since the main purpose of the recrystallization is to decrease the rateof one single identified impurity,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone solubilities weremeasured in a couple of probable antisolvents identified in Table 9. Areference of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone (CHGP059-038) was synthesized at lab on 8 g for this study, with 98% purity,as per International Patent Application No. WO/2005/120584, which isincorporated herein in its entirety. Table 10 below reports thesolubility of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone invarious antisolvent candidates. It is noteworthy that4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is very soluble inacetic acid. As acetic acid is a potential antisolvent to crystallizerofecoxib (RXB-201) (solubility=5 g/L), and it seems to be a goodcandidate that should allow a good crystallization of rofccoxib(RXB-201) while keeping in solution the4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. Alcohols canalternatively be employed as antisolvent.

TABLE 10 Solubility of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone in various solvents Temperature Solubility Solvent(° C.) (g/L) Data Acetic acid 30 1540 Measured n-Butanol 30 14 MeasuredEthanol 30 28 Measured IPA 30 0.2 Measured Water 30 0.7 MeasuredRecrystallization Trials with Various Antisolvents

Isopropanol was tested first as antisolvent, following the processedshown in Tables 11A-B below, LMC P045-157. The recrystallization yieldwas poor, only 64%. Purge factor of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is almost unchangedcompared to the use of water. The low yield is surprising consideringthe low solubility of rofecoxib (RXB-201) in IPA. A mix between IPA andwater was tested as antisolvent (LMC P045-165). This trial led to a muchbetter yield of 93%. while purge of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone stayed around 2.The very high solubility of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone in acetic acidsuggests that this solvent would be a good option to improve theimpurity elimination. Moreover, the low solubility of rofecoxib(RXB-201) should lead to a high yield. LMC P045-076 was launched withacetic acid instead of water. This trial gave a low yield of 68%. with apurge of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone slightlyincreased to 3. This purge factor is far from what was expectedaccording to solubility data. Two final trials were performed with thesame process, but with spiking of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone at the beginning toreach around 1% and 2% of impurity before recrystallization. This led tothe same yield as LMC P045-076, without real effect on purge efficiency.These trials with new antisolvent did not help to significantly improvepurge efficiency of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. Therefore, wateris used as antisolvent.

TABLE 11A Rofecoxib (RXB-201) recrystallization trials with variousantisolvents Crude RXB-201, process RXB- Sulfoxide Reference Scale (%a/a) Solvent Antisolvent LMC P045-157 8 g 7 V DMSO 7.5 V IPA LMCP045-165 8 g 7 V DMSO 1.88 V IPA 5.62 V Water LMC P045-176 8 g 7 V DMSO7.5 V AcOH LMC P045-180-R1 8 g 1.0 7 V DMSO 7.5 V AcOH LMC P045-180-R2 8g 1.9 7 V DMSO 7.5 V AcOh

TABLE 11B Rofecoxib (RXB-201) recrystallization trials with variousantisolvents (cont.) Recrystallized RXB-201 Purge factor UHPLC results(% a/a) (UHPLC) Yield Fura- Sulf- RXB- Fura- Sulf- Reference (%) noneoxide 201 none oxide LMC 64 0.05 0.04 99.89 3.0 2.3 P045-157 LMC 93 0.110.05 99.83 1.4 1.8 P045-165 LMC 68 0.06 0.03 99.83 2.5 3.0 P045-176 LMC66 0.04 0.38 99.32 3.8 2.6 P045-180- R1 LMC 65 n.d. 0.49 99.28 >15 3.8P045-180- R2

TABLE 12A Optimization of rofecoxib (RXB-201) recrystallization withDMSO/water Crude RXB-201, process RXB- Sulfoxide Solvents TemperatureReference Scale (% a/a) DMSO Water Initial Final LMC 8 g 1.1 7 V 7.5 V50° C. 20° C. P045-186- R1 LMC 8 g 1.8 7 V 7.5 V 50° C. 20° C. P045-186-R2 VLA 10.1 g 0.23 5.5 V 5 V 60° C. 20° C. P075-180 VLA 10.1 g 0.22 6 V5.5 V 50° C. 0° C. P075-184 CHG 10.1 g 0.47 5 V 5.5 V 50° C. 0° C.P059-090 CHG 20 g 0.08 5 V 5.5 V 50° C. 20° C. P059-098 CHG 19 g 0.22 5V 3 V 50° C. 20° C. P059-104

TABLE 12B Optimization of rofecoxib (RXB-201) recrystallization withDMSO/water (cont.) Recrystalized RXB-201 UHPLC results (% a/a) Purgefactor (UHPLC) Yield Fura- Sulf- Hy- RXB- Fura- Hy- Sulf- Reference (%)none oxide droxy 201 none droxy oxide LMC 83 0.12 0.45 n.d. 99.34 1.3n.a. 2.4 P045-186- R1 LMC 92 0.12 0.79 n.d. 99.04 1.3 n.a. 2.3 P045-186-R2 VLA 89 n.d. 0.10 0.47 99.43 n.a. 5.6 2.3 P075-180 VLA 92 n.d. 0.110.08 99.81 n.a. 6 2.0 P075-184 CHG 92 n.d. 0.28 0.04 99.67 n.a. 10 1.7P059-090 CHG 97 n.d. 0.05 0.01 99.93 n.a. 2.3 1.6 P059-098 CHG 96 0.050.12 0.02 99.82 n.a. 2 1.8 P059-104

Study of formation of4-[4-(methylsulphonyl)phenyl]-3-phenyl-S-hydroxyfuran-2-one(RXB-Hydroxy) Impurity

There are report of the synthesis of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one under air.This impurity was not detected in pilot batch F801, but is alwaysobserved in trials. In trial VLA P075-180, the rate of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one rose to2.66% at 60° C. before addition of water. The sodium sulfite issuspected to generate this impurity, as depicted in the Scheme 2 below.At pilot, the polishing filtration removes the traces of salt, whilethis filtration is not performed in lab trials. Moreover, the inertatmosphere is better controlled at pilot then at lab.

Trials were performed to check the effect of both temperature and sodiumsulfite on the4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one rate. Purerofecoxib (RXB-201) (F851) was solubilized in 5.5V DMSO. on which Na₂SO₃was added. Three amounts of Na₂SO₃ were tested:

-   -   0%: rofecoxib (RXB-201) post filtration    -   0.5%: ashes result obtained on pilot batch (crude rofecoxib        (RXB-201) F801)    -   1.5%: ashes result obtained on APG P052-110 batch

Samples were subjected to 2 different temperatures under air atmosphere:

-   -   40° C.: current heating temperature for polishing filtration    -   60° C.: possible future temperature for polishing filtration if        DMSO volumes are reduced for better productivity.

UHPLC results are reported in the Table 13 below.

TABLE 13 UHPLC rate of 4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one (RXB- Hydroxy) in function of RoI andtemperature RXB-Hydroxy Residue on (UPLC % a/a) ignition IPC 40° C. 60°C.   0% IPC1 (3 h) 0.08 0.16 IPC2 (6 h) 0.14 0.17 0.5% IPC1 (3 h) 0.300.35 IPC2 (6 h) 0.45 0.52 1.5% IPC1 (3 h) 0.38 0.67 IPC2 (6 h) 0.60 0.73

UHPLC results clearly show that the recrystallization medium (rofecoxib(RXB-201) in 5.5V DMSO) is almost stable at 40° C., and 60° C. for 6hwith an amount of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one up to 0.17%.However, the introduction of sodium sulfite has a negative effect ofpurity profile, as well as increasing temperature. The worst case gavean amount of 4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one9 times higher than at the beginning. The control of the sodium sulfiterate in the crude rofecoxib (RXB-201) is crucial for the UHPLC profileof the recrystallized compound. To solve this issue, the optimizedoxidation process was designed without sodium sulfite quench. Thesuppression of Na₂SO₃ was use tested (Oxidation CHG P059-092 followed byrecrystallization CHG P059-098) and succeeded to give an API that meetsspecifications, as visible in Tables 12A-B above.

Optimization of Recrystallization in DMSO/Water

A. Preliminary Trials

All recrystallization trials conducted with DMSO and water are reportedin Tables 12A-B. The third column“4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone” refers to theamount of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone beforerecrystallization in crude rofecoxib (RXB-201). As most of cruderofecoxib (RXB-201) engaged in recrystallization were clean, spikingswith 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone (CHG P059-038)were performed to evaluate the purge efficiency. Trials LMC P045-186were conducted to evaluate the purge factor of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, with the currentrecrystallization process, with much higher rates of this impurity thanusually (up to 1.8% a/a). Trials R1 and R2 gave the same purge factor(2.4 and 2.3) as usually. This recrystallization process has then thesame purification efficiency, from 0.10% to 1.8% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. The study was thenfocused at reducing total amounts of solvent, to increase productivity.VLA P075-180 was conducted with lower amounts of DMSO (−1.5V) and water(−2.5V) compared to current process. After having performed the standardrecrystallization process with these reduced solvent amounts, theproduct was obtained with 89% yield, with again 2.3 as4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone purge factor.However, the purity of rofecoxib (RXB-201) was not satisfying (99.43%)due to the formation of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one (0.47%).This trial was launched on a crude rofecoxib (RXB-201) which contained ahigh level of Rol (APG P052-110, 1.5% Rol). The high amount ofRXB-Hydroxy was explained by the presence of Na₂SO₃. With higher DMSOrate (6V), and lower temperature (50° C.), the trial VLA P075-184allowed to give rofecoxib (RXB-201) in good yield after filtration at 0°C., with an average purge of4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one and4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

B. Rofecoxib (RXB-201) Solubility Study

To have a precise view of solvents volume reduction capabilities, it wasneeded to know properly the solubility of rofecoxib (RXB-201) dependingon DMSO volumes. Thanks to the Crystal 16 apparatus, solubility andnucleation of rofecoxib (RXB-201) was measured at four differentconcentrations: 3 V, 4 V, 5 V and 6 V. Results are depicted in FIG. 12 .The solubilization curve is reported in blue, and nucleation in orange.The process used for solubilization of RXB-201 at the beginning orrecrystallization process batch F851 is reported as the purple spot(5.5V at 40° C.). To improve the productivity, the concentration can beincreased to 4.5V, with heating at 50° C. (red spot). These conditionsallows a good solubilization of rofecoxib (RXB-201). with a large safetymargin compared to the nucleation temperature (at least 30° C.). Thismargin is required to avoid spontaneous crystallization duringclarifying filtration. An extra-amount of DMSO (+0.5 V) is used forrinsing of the filtration system, leading to 5V DMSO in total.

C. Optimization of Recrystallization Productivity

A recrystallization trial was launched on 20 g of crude rofecoxib(RXB-201) (CHG P059-098) with 5V DMSO in total. 5.5V water were used toensure crystallization, and led to a very high yield (97%) withsatisfying purity. Nevertheless, the crude engaged in recrystallizationwas already pretty clean. A low4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone purge was observedin this case (purge factor=1.6). It has been demonstrated previouslythat the purge factor of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is almost constant(purge factor=2) starting on crude containing 0.1% to 2%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. It is unknownwhether the low purge factor is due to the low amount of impurity toremoved (rupture of purge linearity), the recrystallization conditions(solvents amount and ratio). reproducibility issues or measures ofuncertainty. A last trial (CHG P059-104) was performed with loweredamount of water (3V) to try to improve purging efficiency while keepinghigh yield. The yield stayed high (96%) with 1.8 as purge factor of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, which is in theaverage. 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone is detected inthis batch, at low rate (0.05%). The crude rofecoxib (RXB-201) used inthis trial had the particularity to come from the calorimetry study of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation. This crudecontained traces of 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone and0.22% 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. Therefore,the presence of 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone in thisdemo batch should not be considered as an alert. The solvent ratio (5VDMSO/3V Water) was chosen as the most optimized recrystallizationprocess. This process is the less solvent consuming, and improves theproductivity of this recrystallization step of +45% compared to currentprocess, without affecting the purge efficiency. The slight yellowcoloration of crude rofecoxib (RXB-201) is efficiently removed to afforda white recrystallized rofecoxib (RXB-201) solid.

Flowsheet of optimized recrystallization of rofecoxib (RXB-201) is shownin FIG. 27 .

The solvent screening, assisted by Dynochem software, has highlightedonly 3 candidates as good solvent, and 6 candidates as antisolvent. DMSOwas kept as good solvent for recrystallization. Isopropanol and aceticacid were tried as antisolvent, without success of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone purge improvement.The combination of DMSO and water is still the best option for cruderofecoxib (RXB-201) recrystallization. Optimization has led to thereduction of 45% of the total volume, which has a signification impacton process productivity. The rate of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is almost onlyhalved after recrystallization.

To address this issue, IPC and crude rofecoxib (RXB-201) specificationsas follows in Table 14:

TABLE 14 Oxidation IPC Oxidation IPC and isolated crude RXB-201 Currentspecification New specification RXB-Furanone ≤0.5% <0.15% RXB-Sulfoxide≤0.2% <0.20%

The formation4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one is also nowbetter understood, and should be significantly reduced due to thesuppression of sodium sulfite used for oxidation quench.4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one issufficiently eliminated during recrystallization.

Example 8—Further Process Optimization of Oxidation of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone

Context of the Study

FIG. 28 shows the current flowsheet for4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (RXB-furanone)oxidation.

Optimization was conducted with respect to the following factors:

-   -   The oxidation is classed 5/5 on the Stoessel scale, with high        thermic accumulation phenomenon observed at the beginning of the        oxidation.    -   The time of heating to reach IPC specification        (4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone ≤0.2% area        UHPLC) was 14h instead of 6h. This long reaction time could be        due to a slower reaction or difficulties to get representative        IPC sample of the batch composition. The impurity profile was        not affected, even after this long heating time.    -   A high amount of Rol (0.5%. analysis #CQ18-0487) was obtained in        the isolated crude rofecoxib (RXB-201). This material led to        filter clogging during clarification before recrystallization.

The presence of insoluble matters could be attributed to sodium sulfite,as difficulties were encountered during its dissolution prior to quench.The first part of the optimization work is focused to the research ofsolvents for oxidation process. The second part is focused on thesuppression of energy accumulation to improve the process safety. Theconversion of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone(RXB-Sulfoxide) traces at the end of the oxidation was then studied, aswell as IPC samples preparation. Finally, the need of sodium sulfitequench was evaluated.

Research of Solvents for the Oxidation Process

In the current oxidation process, depicted in Scheme 3, the solvent usedis acetonitrile. In the process conditions, 6 volumes of ACN allow afull dissolution of 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone at65° C. at the beginning of the process. 6h after hydrogen peroxideaddition completion, the reaction medium is heterogeneous, with a highamount of white solid in suspension. This solid is mostly composed ofrofecoxib (RXB-201) (>99%) with traces of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone (<1%). This revealsthat rofecoxib (RXB-201) has a lower solubility in ACN than4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. While rofecoxib(RXB-201) crystallizes out,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone could be stackedinto the crystals. Therefore, a better solubility of RXB species wasresearched to allow a full oxidation of the4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone traces. In the aimof recrystallization improvement, the solubility of rofecoxib (RXB-201)was measured in a large panel of solvents. Before these solubilitieswere measured, several trials were conducted. Scheme 4 below showsoxidation of 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone inDCM/H₂O.

In this trial (CHG P059-058), 6V ACN were substituted by 5V DCM, whichis expected to be a good solvent for polar organic molecules, such asrofecoxib derivatives. Five conditions were studied, as shown in theTable 15 below.

TABLE 15 Screening of conditions for 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanoneoxidation in DCM/H2O. Trial RXB-Furanone Mass (g)H₂O₂ Na₂WO₄•2H₂0 TBACI DCM A F801 dried 2.00 1.55 mL; 23.3 mg; 0 mol %10 mL; 2.5 eq 1 mol % 5 V B VLA P075-132 3.62 1.55 mL; 23.3 mg: 0 mol %10 mL; (PP = 44.8%) 2.5 eq 1 mol % 5 V C VLA P075-132 3.62 1.55 mL: 23.3mg;  98 mg; 10 mL: (PP = 44.8%) 2.5 eq 1 mol % 5 mol % 5 V D VLAP075-132 3.62 1.55 mL; 23.3 mg; 393 mg; 10 mL: (PP = 44.8%) 2.5 eq 1 mol% 20 mol % 5 V E VLA P075-132 3.62 1.55 mL; 23.3 mg: 983 mg: 10 mL: (PP= 44.8%) 2.5 eq 1 mol % 50 mol % 5 V

Trials A and B allowed the study of the effect of residual IPA in4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone on the kinetics. TrialsC, D and E employed a phase transfer catalyst, the tetrabutylammoniumchloride (TBACl). to improve the mixing between organic and aqueouslayers. All trials were run at room temperature. For practicality, 0.5Vof a solution of catalyst (Na₂WO₄.2H₂O) in pure water was introduced.FIG. 13 shows the results of oxidation trials.

Trial A shows a very low conversion of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone with UHPLC. Only 10% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone is observed after3h reaction at room temperature. The influence of IPA content in4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone is visible, with aslightly better conversion (22%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone after 3h at r.t.)obtained in trial B. A dramatic difference was observed with the use ofthe TBACl as phase transfer catalyst. Trials C. D and E show theformation of rofecoxib (RXB-201) as main product, even after 30 minreaction. The three trials do not show a real difference for kinetics.Crystallization occurred during oxidation, suggesting a poor solubilityof rofecoxib (RXB-201) in DCM. Results obtained in DCM are not betterthan with the current process in ACN, considering the lack of conversionand solubility. Scheme 5 below shows oxidation inacetonitrile/sulfolane.

The following trials were conducted in 6V ACN as main solvent. An extraaddition of sulfolane (1V or 2V) was performed to check the effect onsolubility (CHG P059-062). Sulfolane was chosen due to its structurelikeness compared to the rofecoxib (RXB-201). The reaction was launchedon the wet 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone, withcatalyst previously solubilized in water as in biphasic trials as shownin Table 16 below. Results of oxidation trials are reported in FIG. 14 .

TABLE 16 Screening of conditions for 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanoneoxidation with sulfolane. Sul- Trial RXB-FuranoneMass (g) H₂O₂ Na₂WO₄•2H₂0 folane ACN A VLA P075-132 3.62 1.55 mL; 23.3mg; 0 12 mL; (PP = 44.8%) 2.5 eq 1 mol % 6 V B VLA P075-132 3.62 1.55mL; 23.3 mg; 2 mL; 12 mL; (PP = 44.8%) 2.5 eq 1 mol % 1 V 6 V C VLAP075-132 3.62 1.55 mL; 23.3 mg; 4 mL; 12 mL; (PP = 44.8%) 2.5 eq 1 mol %2 V 6 V

As for the biphasic trials, 0.5V of a solution of catalyst (Na₂WO₄.2H₂O)in pure water was introduced, to bring 1 mol % catalyst in the reactionmedium. Then, only 0.25 eq, of H₂O₂ was first introduced to observe apotential energy accumulation. Upon addition of the first drops of H₂O₂in the mixture at 65° C., an exotherm was observed in the 3 reactionmedium. UHPLC confirmed the formation of ≈30% area4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone after 5 min in eachtrials (see FIG. 14 ).

Trial A is equivalent to the current process. Therefore, an addition of0.5V water seems to be efficient to suppress the energy accumulationphenomenon. The rest of the hydrogen peroxide was then introduced andreaction media were let under stirring at 65° C. 30 min after H₂O₂addition, an UHPLC analysis was performed and indicated a totalconversion of 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. However,sulfolane has a negative effect on conversion of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone, with 1.48%remaining intermediate for trial C, 0.98% for trial B. while thestandard process (trial A) shows only 0.23% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. The dilutioneffect may be responsible for such a lack of conversion: 8V in total forC, while 6V in total for A. The use of sulfolane seems to not be veryeffective for improving oxidation of RXB species. Moreover. sulfolanehas a low PDE, the concentration limit in ICH Q3C is 160 ppm. Therefore,the risk-benefit ratio is not favorable for the use of this solvent. The3 solvents below provide rofecoxib (RXB-201) with solubility of morethan 100 g/L at 30° C.:

-   -   DMSO: 107 g/L    -   DMF: 133 g/L    -   NMP: 140 g/L

Being reactive toward the oxidation process, DMSO cannot be used assolvent for the oxidation step. DMF was a solvent widely usedpreviously. This solvent was substituted in all steps due to safetyreasons. Therefore, DMF cannot be considered as an alternative. Finally,NMP revealed to be the best solvent, with a rofecoxib (RXB-201)solubility at 30° C. up to 140 g/L. However, the literature mentions theoxidation of NMP into N-methylsuccinimide with hydrogen peroxide undermetal catalysis at 0° C. See Dong. J. J., et al. Chem Sus Chem, 2013, 6,1-6, which is incorporated herein in its entirety. The processconditions at 65° C. makes it risky to envisage the use of NMP withoutchecking the potential formation of 5-hydroxy-N-methylpyrrolidone andN-methylsuccinimide. Ultimately, NMP was withdrawn from the list ofcandidate solvents for oxidation process. ACN was kept as the solventfor the oxidation process. Addition of water seems to have a positiveeffect on the reaction kinetics as Trial A in FIG. 14 shows only 0.23%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone 30 min afterhydrogen peroxide addition completion. A kinetic model was built tobetter understand the evolution of RXB species over the time. Thethermic accumulation phenomenon of the current process requires aprecise appreciation of the reaction kinetic.

Kinetic Model of RXB Species Oxidation

Previous trials have shown that the use of DCM and sulfolane does notallow to obtain a better conversion. The dissolution of the catalystprior to addition of hydrogen peroxide avoided the accumulationphenomenon. A kinetic model was built to better understand the oxidationreactions that take place in reaction media with the current process.Dynochem® software was used, with the tool “Simple fed batch reaction”to achieve this goal. Two trials were conducted. with the parametersdescribed in Table 17. with reaction follow-up by UHPLC.

TABLE 17 Trials performed for kinetic model building. RXB- H₂O₂ Temper-Furanone 35% Na₂WO₄•2H₂O ACN ature CHG 9.05 g 2.9 eq. 0.012 mol % in 7.1 V 65° C. P059-074 LoD = 53% 0.59 V H₂O CHG 9.05 g 2.9 eq. 0.18 mol% in 28.2 V 45° C. P059-78 LoD = 53% 0.59 V H₂O

To allow for reaction follow up in a reasonable amount of time, trialCHO P059-074 was conducted with an amount of catalyst divided by 100(0.012 mol %). To achieve all kinetic parameters of the reaction, thesecond trial CHG P059-078 was performed at lower temperature (45° C.).To increase the solubility of RXB species in the reaction media, theacetonitrile amount was multiplied by 4 (28.2V). The counterbalance ofthe lower temperature and higher dilution was brought by increasing thecatalyst amount to 0.18 mol %. FIGS. 15A-B show the rate of the speciesin the reaction media over the time. FIG. 15A shows4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation follow-up forCHG P059-074. FIG. 15B shows4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation follow-up forCHG P059-078. Hydrogen peroxide was added in 4 portions (10%, 20%, 30%,and 40%). Each vertical line belongs to an instantaneous addition ofH₂O₂. Instantaneous addition of reagent is mandatory to avoid thelimitation of the reaction kinetic by the addition rate of reagent.Three reactions are considered to take place in the reaction media:

-   -   Na₂WO₄+H₂O₂→Na₂WO₅+H₂O    -   4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone+Na₂WO₅-4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone+Na₂WO₄    -   4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone+Na₂WO₅-RXB-201+Na₂WO₄

The fitting between the model (lines) and the experimental points (dots)were found to be satisfactory. The reaction rate coefficient andactivation energy were determined for the 3 reactions.

The first oxidation(4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone→4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone:k=10 L/mol·s) was revealed to be ten times faster than the second one(4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone→RXB-201: k=1L/mol·s). Regeneration of the catalyst (Na₂WO₄→Na₂WO₅) has a reactionrate constant of k≈8.5 L/mol·s. These data reflect the difficulty tofully convert the 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(51H)-furanoneinto rofecoxib (RXB-201) as the second oxidation is ten times slower.However, these kinetic data have to be cautiously considered in regardto reaction completion. Both oxidations were performed without going toofar in the formation of rofecoxib (RXB-201). This was done on purpose toavoid the crystallization of rofecoxib (RXB-201) at the end of theprocess. Both processes were conducted with fully soluble RXB speciesall along the data acquisition, which simplified the model. This modelshows again the start of oxidation immediately after the addition ofH₂O₂, suggesting a highly decreased energy accumulation. Both trialswere performed with addition of 0.5V water with the catalyst. Thisaddition leads to a diminution of solubility of RXB species in theoxidation media, as RXB species are almost insoluble in water.Therefore, the volume of water was studied to introduce the minimumamount needed for energy accumulation suppression, with the lowestimpact possible on RXB solubilities. Kinetic parameters of reactionstaking place during the oxidation process are shown in Table 18 below.

TABLE 18 Kinetic parameters of reactions taking place during theoxidation process Reactions Phase/ Flow Reaction Equation Rate Exp. krefTref Ea Solution rxn1 cata-red + H202 --> k [cata-red] k> 8,492 L/mol ·s 65.0 C. Ea> 59,604 kJ/mol cata ÷ water [H2O2] Solution rxn2 cata ÷Step 3 --> k [cata] k> 10,169 L/mol · s 65.0 C. Ea> 62,736 kJ/molcata-red ÷ Sulfoxide [Step 3] Solution rxn3 cata ÷ Sulfoxide --> k[cata] k> 1,038 L/mol · s 65.0 C. Ea> 27,446 kJ/mol cata-red ÷ Step 4[Sulfoxide]Optimization of SolventsA. Research of Optimum Water and IPA Contents to Suppress AccumulationPhenomenon

Trials were performed to evaluate the appropriate amount of water tointroduce at the beginning of the reaction to solubilize the catalystand suppress the accumulation phenomenon. Trials below were performedusing the current process in 6V ACN, with dry4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone, catalyst, addition ofIPA to represent different values of LoD and addition of water. Startingthis trial, catalyst and water were successively introduced in thereaction media. The mixture was heated at 65° C., then 0.125 eq H₂O₂were added instantaneously. An IPC was performed after 2 min of contactto check the reaction. IPC results reported in Table 19 below are for4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.

TABLE 19 Screening of IPA and water content to suppress accumulationphenomenon. Pure water 0.15 V 0.25 V  0% VLA P075-168 Slightly cloudedIPC (2 min) = 8.6% IPA 36% VLA P075-166 VIA P075-174 Slightly cloudedSlightly clouded IPC (2 min) = IPC (2 min) = 9% 14.3% 50% VLAP075-170VIA P075-172 Clear + Slightly clouded unsoluble solid IPC (2 min) = IPC(2 min) = 11.7% 4.6%

It is noted that the use of 0.25V of H₂O allows the best conversion into4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone after only 2minutes, up to 14% area UHPLC. When water volume was decreased to 0.15V,the reaction started anyway but the conversion is almost halved. Alltrials have shown a slightly clouded reaction media, except the one withthe maximum amount of IPA and minimum of water (VLA P075-170). Thisindicates a positive effect of IPA and negative effect of water of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone solubility.Nevertheless, a high rate of IPA combined with low rate of watergenerates a different suspension (VLA P075-170), with high-densitycolorless solid, probably insoluble catalyst. It can be assumed that0.25V of pure water allows for a partial dissolution of the catalyst,enough to avoid the accumulation effect. Therefore, this water volumewas chosen. This water amount combines the suppression of theaccumulation effect, with an acceptable solubility of wet4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone for averages LoDs. Theuse of purified water is mandatory. A trial was conducted withdissolution of 12 samples of Na₂WO₄.2H₂O in pure water, and 12 samplesin tap water. The 24 samples were prepared at the same concentration,around working concentration during oxidation synthesis. All samplesdissolved quickly after stirring at room temperature. After few hours,the 12 samples in tap water showed white precipitate while the 12samples in pure water stayed as a clear solution. Calcium cations (Ca2⁺)in tap water are thought to associate with the WO₄ ²⁻ anion to formcalcium tungstate CaWO₄, an almost insoluble salt in water (0.02 g/L).

B. Research into the Effects of Solvents on Oxidation ReactionCompletion.

Previous trials have shown the best conditions to suppress theaccumulation phenomenon. The use of 0.25V of deionized water allowed tostart the reaction at the beginning of hydrogen peroxide addition. Thiswas demonstrated with different values of IPA content in4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone (LoD=36% or 50%). Afterhaving focused on reaction start, the effect of solvents was thenstudied on reaction completion as shown in Table 20 below.

TABLE 20 Effect of Solvents in Reaction Completion. VLA P075-176 VLAP075-178 CHG P059-084 Materials RXB-Furanone 3.00 g 3.00 g 3.00 g H₂O₂35% 2.5 eq. 2.5 eq. 2.5 eq. Na₂WO₄•2H₂O 1 mol % 1 mol % 1 mol % ACN 6 V6 V 7 V IPA 36 % wt. none 36 % wt. H₂O 0.25 V 0.25 V 0.25 V Process H₂O₂addition time 2 h 20 2 h 00 2 h 00 RXB-201 seeding None 1 seed, end of 2seeds during H₂O₂ addition H₂O₂ addition Aspect Slightly clouded WhiteWhite Comments during whole suspension at suspension after process. theend of H₂O₂ first seeding. Addition 1 V addition, before ACN after IPC2:seeding white suspension UHPLC IPC 1 97.23/2.50 99.20/0.53 Not performed(% area) (End of H₂O₂ RXB-201/ addition) Sulfoxide IPC 2 (time)99.72/n.d. 99.68/0.02 99.40/n.d. (1 h) (30 min) (45 min) Work-up Notisolated Not isolated 2 V water Isolated RXB-201 Not isolated Notisolated 99.82/0.02

The first trial (VLA P075-176) was conducted using the standard process(at 65° C.), with addition of 0.25V pure water. Addition of hydrogenperoxide was achieved in 2h. A slightly clouded reaction media wasobtained, with a very low amount of solid in suspension. At the end ofH₂O₂ addition, 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone was notdetected, and only 2.5% E4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone for this trial (IPC1). After an additional hour of stirring at 65° C.,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone was not detected inUHPLC (IPC 2). IPC were performed with sampling of a heterogeneousmixture. This trial is very satisfactory considering the very fastconversion of species into rofecoxib (RXB-201). As a reminder, thecurrent process without water addition needed at least 6 h to reach IPCspecification. At the end of the reaction, 1V ACN was added to thereaction to attempt to achieve a full solubilization of the reactionmedia. In fact, the reaction media turned immediately heterogeneous, aswhite suspension. This indicates an oversaturation of the reactionmedia. Rofecoxib (RXB-201) crystallized out with the perturbationgenerated by the addition of acetonitrile.

The effect of isopropanol content was then studied in the second trial(VLA P075-178). The reaction was performed without IPA. which shouldhave a negative effect of RXB species solubility considering Table 19.In this trial, performed in the same conditions, the reaction media wasalready in suspension at the end of hydrogen peroxide addition. A betterconversion was observed at the end of H₂O₂ addition compared to VLAP075-176. Moreover, the H₂O₂ addition was performed faster than thefirst trial (2 h instead of 2h20). The absence of IPA reveals then afaster oxidation conditions than with 36% IPA. This could be attributedto a slightly higher concentration of reaction media because of theabsence of IPA. The crystallization of the reaction media seems to haveno effect on the 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanoneoxidation kinetics.

A third trial was performed to confirm that point. The trial CHGP059-084 was launched in 7V ACN with usual amount of IPA. This higherdilution revealed to have no effect on reaction media aspect at thebeginning of oxidation. Two seedings were performed during H₂O₂ additionto force the reaction media to be in suspension while addition. Reactionmedia aspect was similar to VLA P075-178, 45 minutes after hydrogenperoxide addition completion,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone was not detected.After addition of 2V water as quench, cooling to 0° C. filtration andcake washings, the solid obtained contained 0.02%4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone. This trialconfirms the satisfactory results obtained in VLA P075-178.

Therefore, the reaction media could be both slightly clouded or aheterogeneous white suspension, the IPC specification was achieved afteronly 1 h of heating after hydrogen peroxide addition completion. Theoversaturation of the reaction media with 6V ACN seems to not be anissue for conversion of RXB species into rofecoxib (RXB-201). The 0.25Vpure water addition has a dramatic effect on oxidation kineticimprovement. The oversaturation of RXB species in oxidation media leadsto the crystallization of samples dedicated to IPCs. A study wasconducted to establish the best sample preparation.

Optimization of IPC Sample Preparation

IPCs were performed by direct sampling of stirred reaction mixture. Thisheterogeneous sampling was difficult to perform representatively.Therefore, the stirred suspension sampling was replaced. The suspendedaliquot was filtrated, and the solid analyzed. Due to the low solubilityof RXB species, it was assumed that the cake analysis representativenesswas satisfactory, particularly in regard to the4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone rate. Thisassumption was verified in this work. A study of IPC preparation wasperformed during trial CHG P059-084 and shown in Table 21 below.

TABLE 21 Screening of conditions of IPC sample preparations. RXB- RXB-RXB- Fura- Sulf- Hy- RXB- Time Temp. H₂O Phase none oxide droxy 201 IPC165° C. 0 V Represen- n.d. 7.90 0.44 91.66 tative IPC2 65° C. 0 VRepresen- n.d. n.d. 0.58 99.40 tative IPC3a 20° C. 0 V Unwashed n.d.0.02 0.09 99.86 cake ML n.d. 0.07 2.13 96.91 IPC3b 20° C. 2 V Unwashedn.d. 0.02 0.12 99.82 cake ML n.d. 0.09 4.39 93.57 Isolated 0° C. 2 VWashed n.d. 0.02 0.07 99.85 cake ML n.d. 0.13 7.85 88.80

The two first IPCs (IPC1 and IPC2) were performed with representativesampling (taking a sample in a stirred suspension). The IPC2 indicated afull conversion:4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone=n.d. IPC3a wasperformed with sampling of 1 mL reaction media, cooling to roomtemperature and filtration. Cake and mother liquors were analyzed,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone=0.02% in the cake.IPC3b was performed with sampling of ≈1 mL reaction media, cooling toroom temperature and addition of ≈2V water and filtration. Cake andmother liquors were analyzed,4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone=0.02% in the cake.The whole product of the experiment was obtained after cooling thereaction media to room temperature, addition of 2V water, cooling to 0°C. filtration and standard cake washings.4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone=0.02% in the cake.

These trials reveal that IPC 3a and IPC 3b techniques give the sameresults of 4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone rate asthe isolated compound of the trial CHG P059-084. If we consider all RXBderivatives. IPC3a technique give the closest results. Another impuritywas reported in the Table 21. called4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one(RXB-Hydroxy). Its proposed structure is shown in FIG. 16 . Thisimpurity is detected at RRT 0.63. LCMS analysis performed duringpre-degradation tests has shown a mass equivalent to [RXB-201+16]. Thishydroxylation is reported in the literature, notably with oxygen. See D.A. Nicoll-Griffith et al. Bioorg. Med. Chem. Lett. 2000, 10, 2683 and E.J. Corey et al. Tetrahedron Lett. 2005, 46, 927. which are incorporatedherein in their entireties. This impurity is always observed inrofecoxib (RXB-201) samples, where nitrogen flow was not used duringoxidation process.4-[4-(methylsulphonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one rate wasonly 0.03% in the crude rofecoxib (RXB-201), batch F801. This lower ratecould be explained by the nitrogen atmosphere used.

Suppression of Sodium Sulfite Quench

In the current synthesis process depicted in Scheme 3, sodium sulfite isused as water solution to quench the excess of hydrogen peroxide addedin the reaction media. Crude rofecoxib (RXB-201) F801 was previouslyobtained with 0.5% residue on ignition. This material is thought to haveled to filter clogging during clarification before recrystallization.The effect of ashes during recrystallization process was also studied.The suppression of sodium sulfite quench was envisaged and use-tested.This quench was replaced by an addition of 2V pure water (CHG P059-092).This trial has shown many advantages:

-   -   A better yield was obtained without Na₂SO₃ use: 92% instead of        86%    -   No effect encountered on crude rofecoxib (RXB-201) UHPLC        profile.    -   No particles in suspension during hot filtration when engaged in        recrystallization (CHG P059-098)

Several verifications were performed to confirm the feasibility ofhydrogen peroxide removal:

-   -   No corrosion of Stainless steel 316L in contact with crude        rofecoxib (RXB-201) mother liquors (enriched in excess hydrogen        peroxide) after 10 days at 20° C. (CHG P059-084).    -   Crude rofecoxib (RXB-201) does not need to be dried.    -   Strip peroxide test negative for both crude and recrystallized        rofecoxib (RXB-201) (CHG P059-098). Crude washings seem to be        efficient enough to remove peroxide traces in cake washings.    -   Stability of isolated crude rofecoxib (RXB-201) towards hydrogen        peroxide is very satisfying. Crude unwashed rofecoxib (RXB-201)        was stored at room temperature for 17 days. No evolution of        UHPLC profile was observed (CHG P059-104).

Considering all these advantages, it was decided to replace the sodiumsulfite quench by an addition of 2V pure water.

Flowsheet of Optimized Oxidation of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone

FIG. 29 shows an optimized flowsheet of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation.

Calorimetry Study

This optimized process depicted in Scheme 6 was subjected to acalorimetry study. The thermal study was performed on Algochem ARLA FDreactor apparatus on 40 g4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone. The results of thecalorimetry, focused on 4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanoneoxidation, are summed up in the Table 22 below.

TABLE 22 Calorimetric data of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone oxidation withoptimized process. sym- step 1:addition bols description unit of H₂O₂ nmol number of step3 mol 0.14 m reaction weight after addition g 285.4 madded reactant weight g 32.3 Tp process temperature ° C. 65 Daddaddition time min 120 Cp average specific heat J/g/° C. 2.3 tfin End ofthermal power after the addition min 60 Q released energy (if exotherm,negative kJ −53.5 result) ΔHr reaction enthalpy by mol of Hyox (ifkJ/mol −382.1 exotherm, negative result) Qr reaction heat by kg of thetotal reaction kJ/kg −187.5 mixture (if exotherm, negative result) Qrreaction heat by kg of added reactant (if kJ/kg −1656.3 exotherm,negative result) Pmax maximum observed power W/kg 52 Pr average observedpower W/kg 32 Acc maximum accumulation of xx % of the % 6% after maxaddition 20% added Acc Accumulation at the end of the addition % 5% fingas release Y/N N Δtad potential adiabatic temperature rise ° C. 81.5 Tfmaximum final temperature with 100% ° C. 146.5 batch accumulation MTSRmaximum final temperature with max ° C. 70.2 max observed accumulation

The reaction enthalpy was measured at ΔHr=−187 kJ/kg with Cp=2.3 J/g/°C. This leads to a potential temperature rise under adiabatic conditionsΔTad=82° C. The exothermal properties of the reaction was already known,but the main improvement of the new process is the maximum ofaccumulation, now measured at 6% after 20% hydrogen peroxide added. Thisaccumulation is divided by 3 compared to the former process. Therefore,the exothermy is well controlled by the addition of hydrogen peroxide.After having taken in account all calorimetric parameters, the rank ofreaction is class 4 over 5 according to Stoessel Scale.

This optimization work has led to the implementation of 0.25V pure wateraddition at the beginning of the process. Due to this water addition,the thermic accumulation phenomenon was dramatically decreased.Furthermore, this addition revealed to have a very positive effect onRXB species oxidation. The IPC specification is now reached after 1 or 2hours after hydrogen peroxide addition completion, instead of 6h. For abetter reproducibility of IPC results, a new technique of IPCpreparation was also tested. The kinetic model has allowed for thebetter understanding of the evolution of oxidation. The oxidation startsfrom the beginning of hydrogen peroxide addition, with a first oxidationten times faster than the second one. The removal of this sodium sulfitequench is validated after having checked all advantages it brings.

Thermal Study Report

The reaction scheme is shown in Scheme 7 below:

The raw materials are shown in Table 23.

TABLE 23 Raw Materials Mass Den- Volume BATCH MW mol Nb Eq Nb (g) sity(mL) RXB furanone 282.36 1.417E−1 1.000 40.0 1 40.0 st3 dry (VLAP072-132) isopropyl 60 2.333E−1 1.646 14 0.785 17.8 alcohol (Aldrich)acetonitrile 41 4.600E+0 32.46 188.6 0.786 239.9 (carlo erba) DI Water18 5.556E−1 3.921 10 1 10.0 sodium 329.9 1.394E−3 0.010 0.46 3.2 0.14tungstate dihydrate (alfa) hydrogen 34 3.541E−1 2.499 32.3 1.135 38.5peroxide 37.3%

A flowsheet for the calorimetric test is shown in FIG. 30 .

Calorimetric data are shown in Table 24 below.

TABLE 24 Calorimetric Data step 1:addition symbols description unit ofH₂O₂ n mol number of step3 mol 0.14 m reaction weight after addition g285.4 m added reactant weight g 32.3 Tp process temperature ° C. 65 Daddaddition time min 120 Cp average specific heat J/g/° C. 2.3 tfin End ofthermal power after the addition min 60 Q released energy (if exotherm,negative kJ −53.5 result) ΔHr reaction enthalpy by mol of Hyox (ifkJ/mol −382.1 exotherm, negative result) Qr reaction heat by kg of thetotal reaction kJ/kg −187.5 mixture (if exotherm, negative result) Qrreaction heat by kg of added reactant (if kJ/kg −1656.3 exotherm,negative result) Pmax maximum observed power W/kg 52 Pr Average observedpower W/kg 32 Acc max maximum accumulation of xx % of the % 6% afteraddition 20% added Acc fin Accumulation at the end of the addition % 5%gas release Y/N N Δtad potential adiabatic temperature rise ° C. 81.5 Tfbatch maximum final temperature with 100% ° C. 146.5 accumulation MTSRmaximum final temperature with max ° C. 70.2 max observed accumulation

Thermal stability data are shown in Table 25 below (DSC).

TABLE 25 Thermal stability DESCRIP- REFER- TYPE TION ENCE THERMALPHENOMENON KINETIC ENERGY Raw Sodium alfa 1: endotherm from 87 ° C. to130 ° C. fast 75 J/g material tungstate 2: exotherm from 236 ° C. to 305° C. slow −120 J/g Raw H₂O₂ 35% acros 1: exotherm from 47 ° C. to 149 °C. fast −981 J/g material 2: endotherm from 275 ° C. to 378 ° C. slow226 J/g raw Stade 3 SHD 390 1: endotherm from 141 ° C. to 155 ° C. fast114 J/g material 173 reaction ACN + SHD 1: exotherm from 79 ° C. to 138° C. slow −33 J/g mixture H₂O₂ 2: exotherm from 174 ° C. to 212 ° C.fast −42 J/g 3: exotherm from 272 ° C. to 371 ° C. fast −492 J/greaction before H₂O₂ If 19 05 1: endotherm from 234 ° C. to 284 ° C.slow 28 J/g mixture addition 08 2: exotherm from 284 ° C. to 362 ° C.slow −32 J/g reaction after H₂O₂ If 19 05 1: exotherm from 308 ° C. to384 ° C. slow −28 J/g mixture addition +3 08 h at 65° C. crude isolated,If 19 05 1: endotherm from 189 ° C. to 214 ° C. fast 93 J/g product wet,not 08 washed 2: exotherm from 339 ° C. to 400 ° C. fast −260 J/g

The addition of H₂O₂ is exothermal and the energy release is wellcontrolled by the rate of H₂O₂ addition (the maximum heat accumulationis 6% after around 20% added). The reaction mixture is homogeneous andstays easily stirable. In DSC, H₂O₂ exhibits an important exotherm from47° C. (−981/g). The mixture acetonitrile/H₂O₂ is unstable from 272° C.(−492 J/g). The reaction mixture before and after addition are morestable with very small exotherms starting at 284° C. Under normalsynthesis conditions, the semi batch process is safe. The MSTR is 70° C.and a TD24 of 144° C. can be calculated. The risk of triggering adecomposition is low but the rate of addition must be well controlledand the reaction has to be correctly triggered to avoid H₂O₂accumulation.

TABLE 26 Additional Data symbols description unit result Tf batchmaximum final temperature with 100% ° C. 146 accumulation Tdec lowestdecomposition temperature by DSC ° C. 47 TD₂₄ Temperature for TMR = 24 h° C. non disponible TD₈ Temperature for TMR = 8 ° C. non disponible Ebboiling point of the reaction mixture ° C. 82 Tp process temperature °C. 65 main reaction heat potential average secondary reaction heatpotential high step ranking 4

The energy of the main reaction can increase the mass temperature to146° C. At this temperature, the decomposition which could be triggeredis the H₂O₂ decomposition, giving an extra energy of 981 J/g of H₂O₂. Ifwe consider that half H₂O₂ decomposes and the other half is used for thereaction, the adiabatic temperature rise would be 24° C., and the finaltemperature 170° C. The decomposition of the reaction mixture should notbe initiated. If a closed vessel is used, the pressure could reach 9bars. If an open vessel is used, without condenser, the boiling pointwould be reached. The whole energy could vaporize 72 g i.e. 38% of theinitial amount of acetonitrile. The power of the reaction can beestimated at ISOW/kg a 82° C. The vapor rate is calculated at around 230m³ of ACN/h. The risk of flooding is likely. The safety of the processdepends on the respect of the temperature and time of H₂O₂ addition.Additional data are shown in Table 26.

FIG. 17 shows a general scheme of the oxidation reaction. FIG. 18 showsa focus on H₂O₂ addition. FIG. 19 shows maximum temperature attainableby the synthesis reaction (TSR). FIG. 20 shows the conversion toreaction product. FIG. 21 shows oxidation reaction power.

Table 27 shows the values used for the classification of the reaction.The reaction classification is also shown in FIG. 22 .

TABLE 27 Values used for the classification of the reaction −ΔH ΔT ad.TMRad Reaction type (J/g) (° C.) (h) Violent exothermicdecomposition >800 400 High reaction (high risk) 400-800 200-400  <8Average reaction (average risk) 100-400  50-200 8-24 Current reaction<100 <50 >24 (low risk in absence of pressure increase)

Example 9—Computational Mutagenicity Analysis of Rofecoxib(TRM-201)-Related Impurities

Introduction

The subject matter described herein also relates to an evaluation of thepotential mutagenicity of impurities of rofecoxib (TRM-201). Theevaluation is performed by testing the structures in several differentin silico software programs, followed by expert review of the in silicodata.

Over the last several years, use of in silico (computational) tools topredict toxicity has increased significantly and has now becomewell-established not only in the pharmaceutical industry, but also inthe chemical and cosmetic space. This is specifically reflected in thearea of potential genotoxic impurities and finalization in 2015 of theInternational Council for Harmonization of Technical Requirements forPharmaceuticals for Human Use (ICH) M7 guideline, which is the firstregulatory document that supports use of in silico tools as an initialsurrogate for conducting in vitro or in vivo testing (ICH M7_Step 5,2015). The purpose of the M7 guidance is to aid in the identificationand characterization of impurities with mutagenic risk and to outlinethe control strategy for the various classes (Class 1-5) of compounds inorder to limit potential carcinogenic risk to subjects. According to ICHM7. “the absence of structural alerts from two complimentaryquantitative structure activity relationship [(Q)SAR] methodologies(expert rule-based and statistical) is sufficient to conclude that theimpurity is of no mutagenic concern, and no further testing isrecommended (Class 5 in FIG. 23 )”.

In silico software programs to predict toxicity have combined biologyand chemistry with modeling and computational science in order toincrease the predictive power in the field of toxicology. In silicotechniques that employ knowledge-based expert systems like DEREK Nexus(Lhasa. Ltd.). Leadscope Expert Alerts (Leadscope, Inc.), and GT_Expert(Multicase, Inc.) are based upon the presence of structural rules.Expert knowledge based on toxicity data and mechanisms is used to createrules on the likelihood of toxicity in a structure, with informationfrom all applicable rules used to make an overall prediction. Rules areusually encoded as one or more substructures that are matched againstthe test chemical. A prediction is made when a predictive alert matches.These rules often indicate a mechanistic basis for any positiveprediction. Statistical-based systems like Model Applier (Leadscope,Inc.). Case Ultra (Multicase, Inc.) and EPA T.E.S.T. (US EPA) are oftenreferred to as Quantitative Structure-Activity Relationship (QSAR)models and are used to predict various toxicity endpoints based on thechemical structure. These models are constructed from historicallaboratory data (training set) where chemical substructures andmolecular properties (descriptors) are generated from the list ofchemicals. Statistically-based mathematical models are built using thesedescriptors to predict the target toxicological effect.

The primary endpoint of concern in the ICH M7 guidance is DNA reactivemutagenicity, with the Ames bacterial mutagenicity assay being thepreferred assay for this endpoint. The structure based in silicoevaluations have good ability to differentiate mutagens fromnon-mutagens with generally high concordance when compared with Amesassay results (Sutter et al. 2013). Genotoxicants that are non-mutagenictypically have threshold mechanisms and usually do not pose carcinogenicrisk to subjects at levels generally present as impurities (EMEAGuideline on the Limits of Genotoxic Impurities, 2006).

Materials and Methods

The current ICH M7 guideline (ICH M7(R1), 2018) states that twocomplimentary in silica methodologies should be used to qualify certaindrug impurities as not mutagenic. To satisfy the ICH M7 guidance, thisreport encompasses in silico analyses using a rule-based system (DEREKNexus) and a statistical-based system (Leadscope Model Applier). Thepredictive data from each of the in silico software programs wasreviewed in order to provide additional supportive evidence on therelevance of any positive or negative predictions and to elucidate anyunderlying reasons for conflicting results. Dobo et al. (2012) havedemonstrated a 94% negative predictive value across industry to predictnegative results using in silico methods, and this value was increasedto 99% when an expert review was also conducted. Following a review ofthe data, an overall mutagenicity prediction was made (positive ornegative).

For recommended control actions, the results were used to classify thestructure from 1-5 according to the ICH M7 control strategy originallydeveloped by Muller et al. (2006). For example, if an impurity is foundto have no structural alerts or has an alert that is concluded to havesufficient data to demonstrate a lack of mutagenicity, then the impuritycan be treated as Class 5 (non-mutagenic). If the impurity has an alertbut the alert is the same as the non-mutagenic parent, or if the alertcan be scientifically dismissed, it can also be considered non-mutagenic(Class 4). If the impurity has an alerting structure that cannot beruled out for potential mutagenicity, the impurity will be consideredClass 3 and should be controlled at or below the generic threshold oftoxicological concern (TTC) value of 1.5 μg/day or the adjusted TTC,based on duration of dosing.

In Silico Software—DEREK Nexus

DEREK is a knowledge and rule based predictive toxicology softwareprogram that makes qualitative estimations of endpoint risk. A knowledgebased system is a computer program that contains expert knowledge rulesin toxicology and applies the rules to make predictions about thetoxicity of chemicals, usually when no experimental data are available.The qualitative estimations of risk are categorized, in descending orderof probability, as ‘certain’, ‘probable’. ‘plausible’. ‘equivocal’‘doubted’. ‘improbable’. ‘impossible’ or ‘inactive’.

DEREK Nexus contains expert derived functionality to provide negative(inactive) predictions for bacterial in vitro mutagenicity. The LhasaAmes reference set was composed of a variety of data sets (e.g., NTP,FDA CFSAN, ISSSTY, Kirkland, Bursi, Benchmark, Acid Halide Data, MemberData, etc.) and is comprised of 9,900 compounds with 132 mutagenicityalerts (Lhasa Knowledge Suite, Nexus 2.2 Release Notes).

Non-alerting compounds are evaluated to identify unclassified andmisclassified features. Misclassified features in the molecule arederived from non-alerting mutagens in the Lhasa reference set. Featuresin the molecule that are not found in the Lhasa reference set areconsidered unclassified. For compounds where all features in themolecule are found in accurately classified compounds from the referenceset, a negative prediction (inactive) is displayed. Predictions forcompounds with misclassified or unclassified features remain negative,and these features are highlighted to enable expert assessment of theprediction. Negative predictivity has been reported to be high (86-94%),comparable to the Ames assay, for compounds with no misclassified orunclassified features.

The DEREK analysis was conducted specifically using the mutagenicityendpoint.

In Silico Software—CASE Ultra

CASE Ultra (CASE) software is a statistical (GT1_BMut model) andrule-based (GT_Expert) system designed to uncover the relationshipbetween the structure of the chemicals and their activity in a specificbiological assay. It has been designed to deal with “non-congeneric”databases, that is, databases consisting of structurally unrelatedmolecules that are not normally amenable to treatment with traditionalQuantitative Structure-Activity Relationship (QSAR) type techniques. Assuch, its main goal is to find the structural entities that discriminateactive molecules from the inactive ones and its success is dependent onthe validity of the working hypothesis that a relationship does indeedexist between chemical structure and activity. As indicated above, theprogram selects its own descriptors from a number of possiblesubstructural units and creates a dictionary of molecular descriptorswithout human intervention. The selected descriptors are characterizedeither as activating (biophore) or as inactivating (biophobe). Theprogram also considers several other factors, such as molecular weight,octanol/water Log P. water solubility. Lipinski's Rule of 5, andintestinal absorption. All of the alert contributions are considered anda scaled alert weight and regression coefficient are used in the finaloverall probability calculation. Unknown fragments, positive(activating) and deactivating alerts are highlighted in the program, andan overall positive probability is also provided. The ability of CASEUltra to select alerts that are readily recognized as being part of amolecule is a major advantage of the method. Indeed, the identificationby CASE Ultra of structural components embedded in the molecule offers afoothold that human intelligence can exploit with respect to thepossible structural site of metabolism or receptor binding. The moduleswere developed through a collaborative effort between the U.S. FDA andMulticase. Inc. The database (version 3.0) contains 13.514 uniquestructures (6982 positives, 6532 negatives).

Konsolidator is a knowledge driven algorithm that generates usefulsupporting evidence in order to assist those performing expert review.It generates supporting evidence required for expert review andregulatory submissions of in silico predictions. It takes test resultsfrom multiple statistical and expert rule models and re-evaluates thealerts by running queries with a large database of chemicals.Konsolidator was introduced with CASE Ultra version 1.6.0.0 and atpresent only supports bacterial mutagenicity models.

Model Applier

Leadscope® FDA Model Applier (LSMA) is a statistical-based system thatuses (Q)SAR models to provide a quantitative predictive probability forthe potential toxicity of chemicals. All of the Model Applier (Q)SARswere constructed at the FDA by the Informatics and Computational SafetyAnalysis Staff (ICSAS). Complete documentation of the weight of evidencemethodology used for the preparation of the model training sets and thesources of the data have been published by the ICSAS group (Matthews etal., 2008).

The LSMA assessment was conducted with domain analysis using the ICH M7settings. The settings for this application specify a PredictionProbability under 0.4 to be negative and a probability of greater thanor equal to 0.6 to be positive. If the Prediction indicates“Indeterminate” then the probability fell between those cutoffs. Foranalysis purposes in this report, prediction probability scores of20.61-0.79 are considered moderately positive and 0.80-1.0 areconsidered strongly positive.

The Bacterial Mut model is new in the 2018 software. It was constructedto incorporate data from the SAR Genetox database. Bacterial Mutationalerts reference set and the existing FDA RCA models (Salmonella Mut andE coli—TA 102 A-T Mut) in order to improve performance across the board.The training set contains 9109 training compounds (4710 positive/3752negative). The performance metrics for the model (version 1.0) whentested against itself and cross-validated are as follows:

Cross-validated Actual (5% LMO) 83.4 Accuracy 84.6 82.9 Sensitivity 84.184.3 Specificity 85.3 86.6 Positive Predictivity 87.8 79.9 NegativePredictivity 81.0

For all structures, the Model Applier analysis was conductedspecifically using the mutagenicity endpoint.

EPA T.E.S.T.

The EPA Toxicity Estimation Software Tool (TEST) has been developed bythe Environmental Protection Agency to allow users to easily estimatetoxicity using a variety of QSAR methodologies. TEST provides multipleprediction methodologies (hierarchal, FDA. single model, groupcontribution, nearest neighbor, consensus and random forest) so that onecan have greater confidence in the predicted toxicities. Severalendpoints are available for evaluation; however, only the Amesmutagenicity endpoint was used in this evaluation. The predictedtoxicity from the consensus method represents the average of thepredicted toxicities from all the different QSAR methods incorporatedinto the TEST software. The consensus method achieved the bestprediction accuracy (concordance) and prediction coverage for the Amesassay, and this was the method used for the current evaluation. In TEST.predicted values ≥0.50 are considered positive. For analysis purposes,prediction probability scores of ≥0.61-0.79 are considered moderatelypositive and ≥0.80-1.0 are considered strongly positive.

Molecular descriptors (physical characteristics of the structure) werecalculated using computer code written in Java. The basis of themolecular calculations was the Chemistry Development Kit (Steinbeck etal., 2003). The descriptor values were validated using MDL QSAR, Dragon,and Molconn-z. The descriptor values were generally in good agreement(aside from small differences in the descriptor definitions fordescriptors such as the number of hydrogen bond acceptors). The finaldataset consists of 5743 chemicals, which were based of the datasetcompiled by Hansen et al. (2009).

Rofecoxib (TRM-201)

Per NDA 21,042 (1999) and the drug label for VIOXX® (rofecoxib tabletsand oral suspension) (2016):

Rofecoxib (L-748,731) was tested in a battery of genotoxicity assays andwas not found to be mutagenic or elastogenic. L-748,731 was negative formutagenicity in Salmonella typhimurium (TA98, TA100, TA1535, TA97a) andE. coli (WP2, WP2uvrA, WP2 uvrA pKM101) in the presence and absence ofan exogenous metabolic activation system (S9) when tested up to 6000μg/plate. L-748,731 was negative in the in vitro assay for mutations inChinese hamster lung cells at all concentrations tested with and withoutS-9. In the chromosomal aberration study conducted using CHO cells,there were no significant increases in the percent of aberrant cells at25 to 125 μM with S-9 or 25 to 100 μM without S-9 activation.

Rofecoxib was also not carcinogenic in mice or rats at dose levels up to60 mg/kg and 8 mg/kg, respectively.

Structures Evaluated 1. A-RSM1-00 Name:1-(4-(ethylthio)phenyl)ethan-1-one Molecular Weight: 180.27 g/mol

2. A-RSM1-01 Name: ethyl(phenyl)sulfane Molecular Weight: 138.23 g/mol

3. A-RSM1-02 Name: 1-(2-(methylthio)phenyl)ethan-1-one Molecular Weight:166.24 g/mol

4. A-RSM2-00 Name: 2-phenylacetic acid Molecular Weight: 136.15 g/mol

5. A-RSM2-01 Name: 2-phenylacetonitrile Molecular Weight: 117.15 g/mol

6. A-RSM2-02 Name: 2-phenylacetamide Molecular Weight: 135.17 g/mol

7. A-CRM1-00 Name: tetrabutylammonium Molecular Weight: 239.17 g/mol

8. A-STG1-00 Name: 2-bromo-1-(4-(methylthio)phenyl)ethan-1-one MolecularWeight: 245.13 g/mol

9. A-STG1-01 Name: 2,2-dibromo-1-(4-(methylthio)phenyl)ethan-1-oneMolecular Weight: 324.03 g/mol

10. A-STG2-00 Name: 2-(4-(methylthio)phenyl)-2-oxoethyl 2-phenylacetateMolecular Weight: 300.37 g/mol

11. A-STG2-01 Name: 1-bromo-2-(4-(methylthio)phenyl)-2-oxoethyl2-phenylacetate Molecular Weight: 379.27 g/mol

12. A-STG3-00 Name: 4-(4-(methylthio)phenyl)-3-phenylfuran-2(5H)-oneMolecular Weight: 282.36 g/mol

13. A-STG3-01 Name:5-hydroxy-4-(4-(methylthio)phenyl)-3-phenylfuran-2(5H)-one MolecularWeight: 298.36 g/mol

14. A-STG4-01 Name: 4-(4-(methylsulfinyl)phenyl)-3-phenylfuran-2(5H)-oneMolecular Weight: 298.36 g/mol

15. A-STG4-02 Name:5-hydroxy-4-(4-(methylsulfonyl)phenyl)-3-phenylfuran-2(5H)-one MolecularWeight: 330.35 g/mol

16. A-STG4-03 Name: 3-(4-(methylsulfonyl)phenyl)-4-phenylfuran-2,5-dioneMolecular Weight: 328.34 g/mol

Results

1. A-RSM1-00

In rule-based DEREK. A-RSM1-00 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’. with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier. A-RSM1-00 was predictednegative in the Bacterial Mut model with a probability score of 0.399.Most of the main features of the structure were covered in the model andseveral analogs were shown to support the prediction.

2. A-RSM1-01

In rule-based DEREK, A-RSM1-01 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, a prediction for A-RSM1-0lwas not made because the predictive value was 0.472. which is in thegray zone where a confident prediction cannot be made by the software.However, non-mutagenic 1,2-bis(phenylthio)ethane (LS-1491) is shown asan analog in the training set, with 79% similarity. Due to theinconclusive prediction in Model Applier, A-RSM1-01 was also evaluatedusing Case Ultra. It was predicted negative in the GT1_BMut model with acalculated probability of 22.9%: no alerts were identified.

3. A-RSM-02

In rule-based DEREK, A-RSM1-02 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, a prediction for A-RSM1-02was not made because the predictive value was 0.4, which is in the grayzone where a confident prediction cannot be made by the software. Due tothe inconclusive prediction in Model Applier. A-RSM1-02 was alsoevaluated using Case Ultra. It was predicted inconclusive in theGT1_BMut model with a calculated probability of 48.3%, which is withinthe gray zone of 40-60%. One positive alert was identified but it wasdismissed by the software because the analogs were negative, and all thepositive analogs contained reactive groups not present in A-RSM1-02. TheKonsolidator overall outcome call was negative because all of theidentified alerts/features were found to be irrelevant to mutagenicactivity.

For further confidence in the statistical-based predictions, A-RSM1-02was analyzed using TEST and it was predicted negative for mutagenicitywith a predicted value of 0.09. The similarity coefficients from theanalogs in the external and training sets were as high as 0.80 andrelevant analogs were shown to support the prediction, includingnon-mutagenic CASRN 123-09-1 (p-Chlorophenyl methyl sulfide) (Leber etal., 1993).

4. A-RSM2-00

In rule-based DEREK. A-RSM2-00 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’. with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier. A-RSM2-00 was predictednegative in the Bacterial Mut model with a probability score of 0.072.The main features of the structure were covered in the model andrelevant analogs were shown to support the prediction, including LS-7536(alpha-Naphthylacetic acid) with 72% similarity.

5. A-RSM2-01

In rule-based DEREK. A-RSM2-01 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, A-RSM2-01 was predictednegative in the Bacterial Mut model with a probability score of 0.145.The main features of the structure were covered in the model andA-RSM2-0l was an exact match in the Leadscope data base with LS-817(phenylacetonitrile). which was experimentally negative formutagenicity.

6. A-RSM2-02

In rule-based DEREK. A-RSM2-02 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’. with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, A-RSM2-02 was predictednegative in the Bacterial Mut model with a probability score of 0.170.The main features of the structure were covered in the model and severalanalogs were shown to support the prediction.

7. A-CRM1-00

In rule-based DEREK, A-CRM1-00 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features. The Br^([III])salt feature was considered as an unclassified feature. Unclassifiedfeatures (are those that were not found in the Lhasa Ames test referenceset and do not match any structural alerts or examples for (bacterial invitro) mutagenicity in Derek. It is predicted to be inactive in thebacterial in vitro (Ames) mutagenicity test.

In statistical-based Leadscope Model Applier. A-CRM1-00 was predictednegative in the Bacterial Mut model with a probability score of 0.055.The main features of the structure were covered in the model andrelevant analogs were shown to support the prediction, includingnon-mutagenic LS-190530 (tetrapropylammonium bromide: CASRN 1941-30-6).

8. A-STG1-00

In rule-based DEREK. A-STG1-00 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’. with no misclassified or unclassified features. While containsan alkyl bromide, the 027 alert in DEREK contains several exclusioncriteria, including one for primary alpha-halo ketones. Studies suggestthat some phenacyl bromides may be oxidized by the DMSO solvent toafford mutagenic phenylglyoxals, which give rise to positive responsesthat are not observed when using acetone as the solvent (Azuma, et al.,1997).

In statistical-based Leadscope Model Applier, A-STG1-00 was predictedpositive in the Bacterial Mut model with a probability score of 0.770.The positive prediction was mainly due to the alkyl bromide feature.While several analogs with an alkyl halide feature were shown to supportthe prediction, analog LS-188087 (2-bromoacetophenone) with 62%similarity was shown and was experimentally negative for mutagenicity,and analog LS-394467 (2-Bromo-1-[4-(methylsulfonyl)phenyl]-1-ethanone),with 54% similarity was experimentally positive for mutagenicity.

For further confidence in the statistical-based predictions. A-STG1-00was analyzed using TEST and it was predicted negative for mutagenicitywith a predicted value of 0.40. The similarity coefficients from theanalogs in the external and training sets were as high as 0.66 andrelevant alkyl halide analogs were shown to support the prediction.

9. A-STG1-01

In rule-based DEREK. A-STG1-01 was predicted plausible for ‘mutagenicityin vitro bacterium (Salmonella typhimurium and Escherichia coli)’ inDEREK due to matched alert 326 for gem-Dihalide (alerting pharmacophorehighlighted below in gray). Compounds in this class have been shown tobe mutagenic in the Ames test, both in the presence and absence ofmetabolic activation and activity has generally been demonstrated forterminal gem-dihalides, gem-dibromides and gem-mixed dihalides which arealpha to an unsaturated carbon atom, for examplealpha,alpha-dichlorotoluene (Zeiger, et al., 1992).

In statistical-based Leadscope Model Applier. A-STG1-01 was predictedpositive in the Bacterial Mut model with a probability score of 0.870.The positive prediction was mainly due to the alkyl dibromide feature,and several analogs with an alkyl bromide feature were shown to supportthe prediction. Although not a dihalide, 2-bromoacetophenone (LS-188087)was shown as an analog and was experimentally negative for mutagenicity.

The mechanism by which gem-dihalides exert their mutagenic effect is notclear but it is likely to involve the direct reaction of these compoundswith DNA since they are inherently electrophilic species. The reactivityof a given gem-dihalide will depend on a number of factors including thesteric and electronic environment surrounding the reactive center andthe nature of the halogen atoms forming the functional group, withdichloro compounds being less reactive than dibromo compounds.

10. A-STG2-00

In rule-based DEREK, A-STG2-00 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, A-STG2-00 was predictednegative in the Bacterial Mut model with a probability score of 0.235.The main features of the structure were covered in the model andrelevant analogs were shown to support the prediction.

11. A-STG2-01

In rule-based DEREK, A-STG2-0l was predicted plausible for ‘mutagenicityin vitro bacterium (Salmonella typhimurium and Escherichia coli)’ inDEREK due to matched alert 027 for an alkylating agent (alertingpharmacophore highlighted below in gray). The 027 alert coversalkylating agents where the carbon bearing the functional group is aprimary or secondary alkyl carbon atom, and it includes alkylsulphinates, sulphonates and sulphates which lack a hydroxyl groupdirectly bonded to the sulphur.

In statistical-based Leadscope Model Applier. A-STG2-01 was predictedpositive in the Bacterial Mut model with a probability score of 0.739.The positive prediction was mainly due to the alkyl bromide feature, andseveral analogs with an alkyl bromide feature were shown to support theprediction.

Alkyl halides are electrophilic species that are capable of directlyalkylating DNA. Shorter chain alkyl chlorides, such as methyl chloride,are known to be mutagenic (Andrews et al., 1976).

12. A-STG3-00

In rule-based DEREK. A-STG3-00 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, a prediction for A-STG3-00was not made because the predictive value was 0.488, which is in thegray zone where a confident prediction cannot be made by the software.However, rofecoxib is shown as an analog in the training set, with 59%similarity.

Due to the inconclusive prediction in Model Applier, A-STG3-00 was alsoevaluated using Case Ultra. It was predicted negative in the GT1_BMutmodel with a calculated probability of 17.8%; 2 alerts and 1deactivating feature were identified. All of the alerts were dismissedby the software because a majority of the analogs were negative, and thepositive analogs contained reactive groups not present in A-STG3-00.Rofecoxib was also shown as an analog in the database, with 90.4%similarity.

13. A-STG3-01

In rule-based DEREK, A-STG3-0l was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, a prediction for A-STG3-01was not made because the predictive value was in the gray zone at 0.486.However, rofecoxib is shown as an analog in the training set, with 43%similarity.

Due to the inconclusive prediction in Model Applier, A-STG3-01 was alsoevaluated using Case Ultra. It was predicted inconclusive in theGT1_BMut model with a calculated probability of 42.4%; 2 alerts wereidentified. All of the alerts were dismissed by the software because amajority of the analogs were negative, and the majority of positiveanalogs contained reactive groups not present in A-STG3-00. Rofecoxibwas also shown as an analog in the database, with 50% similarity. TheKonsolidator suggested outcome is a negative call because thealerts/features were found to be irrelevant to mutagenic activity.

14. A-STG4-01

In rule-based DEREK, A-STG4-01 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, A-STG4-01 was predictednegative in the Bacterial Mut model with a probability score of 0.353.Most of the main features of the structure were covered in the model andrelevant analogs, including non-mutagenic rofecoxib, were shown tosupport the prediction.

15. A-STG4-02

In rule-based DEREK. A-STG4-02 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, A-STG4-02 was predictednegative in the Bacterial Mut model with a probability score of 0.162.The main features of the structure were covered in the model andrelevant analogs, including non-mutagenic rofecoxib, were shown tosupport the prediction.

16. A-STG4-03

In rule-based DEREK. A-STG4-03 was predicted inactive (negative) for‘mutagenicity in vitro bacterium (Salmonella typhimurium and Escherichiacoli)’, with no misclassified or unclassified features.

In statistical-based Leadscope Model Applier, A-STG4-01 was predictednegative in the Bacterial Mut model with a probability score of 0.182.The main features of the structure were covered in the model andrelevant analogs, including non-mutagenic rofccoxib, were shown tosupport the prediction.

The emphasis of the overall computational assessment of toxicity wasplaced on the potential for DNA reactive mutagenicity. A summary of thein silico results and overall mutagenicity predictions are listed belowin FIG. 23 .

CONCLUSIONS

In accordance with ICH M7 criteria, the potential mutagenicity ofimpurities of TRM-201 was evaluated in silico in at least 2complimentary software programs followed by expert review of the data.Individual model results and overall mutagenicity predictions arepresented in FIG. 23 .

A-RSM1-00, A-CRM1-00. A-RSM2-00. A-RSM2-02, and A-STG2-00 were predictedclearly negative in rule-based DEREK and statistical-based ModelApplier. No alerts were identified, and relevant analogs were shown tosupport the predictions. Therefore, according to ICH M7, they can beconsidered non-mutagenic (Class 5).

A-RSM1-01 was predicted clearly negative in rule-based DEREK andstatistical-based Case Ultra. No alerts were identified, and relevantanalogs were shown to support the predictions. Therefore, according toICH M7. it can be considered non-mutagenic (Class 5).

A-RSM1-02 was predicted negative for mutagenicity in rule-based DEREKand in statistical-based methods, it was considered inconclusive in bothModel Applier and Case Ultra but was predicted negative in TEST.Relevant analogs were shown to support the prediction. The negativeprediction and lack of structural alerts suggests A-RSM1-02 isnon-mutagenic. Therefore. it is considered a Class 5 impurity.

A-RSM2-01 was predicted clearly negative in rule-based DEREK andstatistical-based Model Applier. It was an exact match in the Leadscopedatabase, with negative mutagenicity data. Therefore, it is considered aClass 5 impurity.

A-STG1-00 was predicted negative in rule-based DEREK and positive instatistical-based Model Applier due to an alert for the alkyl bromidefeature. The alkyl halide feature did not alert in DEREK due to anexclusion for phenacyl bromides, which may be oxidized by DMSO used inthe Ames assay resulting in false positive results. In the case forA-STG1-00, the sulfur will be electron withdrawing because of itsability to have some double bond character, thus supporting thedecreased reactivity of this structure. A-STG1-00 was also predictednegative in statistical-based TEST. Thus, the evidence suggestsA-STG1-00 is non-mutagenic (Class 4).

A-STG1-01 was predicted positive in rule-based DEREK andstatistical-based Model Applier due to alerts for the alkyl dibromidefeature. While the mechanism by which gem-dihalides exert theirmutagenic effect is not clear, it likely involves a direct interactionwith DNA due to the inherent electrophilicity of these species. Theactual reactivity of any given gem-dihalide depends on a number offactors, and considering A-STG1-01 does not afford much in the way ofsteric hindrance and it contains a dibromide feature, which is typicallymore reactive than a dichloro feature, A-STG1-01 is consideredpotentially mutagenic until tested in an Ames assay (Class 3).

A-STG2-01 was predicted positive in rule-based DEREK andstatistical-based Model Applier due to alerts for the alkyl bromidefeature. Alkyl halides are electrophilic species that are capable ofdirectly alkylating DNA, and with positive predictions in twocomplimentary in silico systems. A-STG2-01 is considered potentiallymutagenic (Class 3).

A-STG3-00 was predicted negative for mutagenicity in rule-based DEREKand in statistical-based Case Ultra. Although a prediction was not madein Model Applier, non-mutagenic rofecoxib was shown as a close analog.A-STG3-00 was predicted negative in two complimentary in silico systemsand is therefore considered non-mutagenic (Class 5).

A-STG3-01 only differs from A-STG3-00 (above) by the presence of ahydroxyl group on the furan ring, as such, the prediction was similar.A-STG3-01 was predicted negative for mutagenicity in rule-based DEREKand in statistical-based methods, it was considered inconclusive in bothModel Applier and Case Ultra. Although a prediction was not made in thestatistical models, non-mutagenic rofecoxib was shown as a close analog.The negative prediction and similarity to the non-mutagenic parentcompound suggests A-STG3-0l is non-mutagenic. Therefore, it isconsidered a Class 5 impurity.

A-STG4-01. A-STG4-02, and A-STG4-03 were predicted clearly negative inrule-based DEREK and statistical-based Model Applier. No alerts wereidentified, and relevant analogs, including rofecoxib, were shown tosupport the predictions. These structures are very structurallysimilarity to the non-mutagenic parent compound, rofecoxib. Therefore,according to ICH M7. they can be considered non-mutagenic (Class 5).

References for Example 9

Andrews A W, Zawistowski E S. Valentine C (1976). A comparison of themutagenic properties of vinyl chloride and methyl chloride. MutationResearch. 40: 273-275.

Case Ultra, version 1.7.0.5., Multicase, Inc. (Beachwood. Ohio).

DEREK Nexus. version 6.0.1 (2018). Nexus v.2.2.1, Lhasa Limited (Leeds,UK).

Dobo K. Greene N. Fred C. et al. (2012). In silico methods combined withexpert knowledge rule out mutagenic potential of pharmaceuticalimpurities: An industry survey. Regul Toxicol Pharmcol., 62(3):449-55.

EMEA/CHMP/QWP/251344/2006 (2006). Guideline on the limits of GenotoxicImpurities.

EPA T.E.S.T., version 4.2.1 (2016). A Program to Estimate Toxicity fromMolecular Structure. U.S. EPA.

Hansen K. Mika S, Schroeter T. et al. (2009). Benchmark Data Set for insilico Prediction of Ames Mutagenicity. Journal of Chemical Informationand Modeling. 49 (9):2077-2081.

ICH M7 Guideline (2015). Assessment and Control of DNA Reactive(Mutagenic) Impurities in Pharmaceuticals to Limit PotentialCarcinogenic Risk. May 2015.

ICH M7(R1) Guideline (2018). Assessment and Control of DNA Reactive(Mutagenic) Impurities in Pharmaceuticals to Limit PotentialCarcinogenic Risk. March 2018.

Leadscope (2018). Model Applier v2.4.1-36, Enterprise v3.7.1-36;Personal v4.7.1-36. LeadScope®, Inc. (Columbus, Ohio).

Leber A. Dacre J. Thake D. et al. (1993). p-Chlorophenyl methyl sulfide,p-Chlorophenyl methyl sulfoxide, and p-Chlorophenyl methyl sulfone—acutetoxicity and bacterial mutagenicity studies. J. Am. Coll. Toxicol.,12(4):369-376.

Matthews E, Kruhlak N. Benz D, Contrera J. (2008). Combined Use ofMC4PC. MDL-QSAR, BioEpisteme, Leadscope PDM, and Derek for WindowsSoftware to Achieve High-Performance, High-Confidence, Mode ofAction-Based Predictions of Chemical Carcinogenesis in Rodents.Toxicology Mechanisms and Methods, 18:189-206.

Müller L. Mauthe R. Riley C. et al. (2006). A rationale for determining,testing, and controlling 724 specific impurities in pharmaceuticals thatpossess potential for genotoxicity. Regul Toxicol Pharmacol. 44:198-211.

NDA 21.042 (1999). VIOXX (rofecoxib). Merck & Co.

Steinbeck C. Han Y, Kuhn S. et al. (2003). The Chemistry Development Kit(CDK): An Open-Source Java Library for Chemo- and Bioinformatics.Journal of Chemical Information and Computer Sciences. 43:493-500.

Sutter A, Amberg A, Boyer S, Brigo A. et al. (2013). Use of in silicosystems and expert knowledge for structure-based assessment ofpotentially mutagenic impurities. Reg. Toxicol. Pharmacol., 67:39-52.

US Food and Drug Administration. VIOXX (Rofecoxib) U.S. PrescribingInformation May 9, 2016.

Zeiger E, Anderson B, Haworth S, Lawlor T and Mortelmans K (1992).Salmonella mutagenicity tests: V. Results from the testing of 311chemicals., Environmental and Molecular Mutagenesis, 19 (supplement 21),2-141.

EQUIVALENTS

The subject matter described herein can be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. The foregoing embodiments are therefore to be considered in allrespects illustrative rather than limiting on the subject matterdescribed herein. Scope of the subject matter described herein is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method for treating arthritis or migraine in asubject, the method comprising administering to the subject once daily apharmaceutical composition comprising 17.5 mg of highly pure rofecoxib,or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, wherein the method results in the subject decreasingor discontinuing the use of opioid medications, wherein the highly purerofecoxib comprises less than 0.10% total impurities.
 2. The method ofclaim 1, wherein the highly pure rofecoxib is free of4-[4-(methylsulfonyl)phenyl]-3-phenyl-2,5-furandione.
 3. The method ofclaim 1, wherein the highly pure rofecoxib comprises less than 0.10% of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone.
 4. The method of claim3, wherein the highly pure rofecoxib comprises less than about 0.05% of4-[4-(methylthio)phenyl]-3-phenyl-2(5H)-furanone.
 5. The method of claim1, wherein the highly pure rofecoxib comprises less than about 0.05% of4-[4-(methylsulfinyl)phenyl]-3-phenyl-2(5H)-furanone.
 6. The method ofclaim 1, wherein the highly pure rofecoxib comprises less than about0.05% of 4-[4-(methylsulfonyl)phenyl]-3-phenyl-5-hydroxyfuran-2-one. 7.The method of claim 1, wherein the migraine is migraine associated withvon Willebrand disease.
 8. The method of claim 1, wherein the migraineis migraine with aura.
 9. The method of claim 1, wherein the migraine ismigraine without aura.
 10. The method of claim 1, wherein the subjecthas a bleeding disorder and the pharmaceutical composition isco-administered with factor replacement therapy.
 11. The method of claim1, wherein the subject has a bleeding disorder and is being administeredor is taking factor replacement therapy prophylactically.
 12. The methodof claim 1, wherein the subject expresses von Willebrand factor at alevel about 50% below normal.
 13. The method of claim 1, wherein thesubject has been diagnosed with hemophilia A or B.
 14. The method ofclaim 1, wherein the subject has factor VIII or factor IX deficiencywith or without inhibitor.
 15. The method of claim 1, wherein thepharmaceutical composition is an oral dosage form.
 16. The method ofclaim 15, wherein the pharmaceutical composition is a solid oral dosageform.
 17. The method of claim 16, wherein the solid oral dosage form isa capsule, tablet, pill, dragée, powder, or granule.
 18. The method ofclaim 15, wherein the pharmaceutical composition is a liquid oral dosageformulation.
 19. The method of claim 18, wherein the liquid dosage formis an emulsion, microemulsion, solution, suspension, syrup, or elixir.20. The method of claim 1, wherein the subject is 12 years old or older.21. The method of claim 1, wherein the subject is between 12 years oldand 75 years old.
 22. The method of claim 1, wherein the treatmentachieves a reduction of at least 1 from baseline in a Pain IntensityNumerical Rating Scale.
 23. The method of claim 22, wherein thetreatment achieves a reduction of at least 2 from baseline in a PainIntensity Numerical Rating Scale.
 24. The method of claim 23, whereinthe treatment achieves a reduction of at least 3 from baseline in a PainIntensity Numerical Rating Scale.
 25. The method of claim 24, whereinthe treatment achieves a reduction of at least 4 from baseline in a PainIntensity Numerical Rating Scale.
 26. The method of claim 25, whereinthe treatment achieves a reduction of at least 5 from baseline in a PainIntensity Numerical Rating Scale.
 27. The method of claim 1, furthercomprising the subject decreasing or discontinuing the use ofacetaminophen during the course of the treatment when compared to beforethe initiation of the treatment.
 28. The method of claim 1, wherein themethod results in the subject decreasing or discontinuing the use ofopioid medications during the course of the treatment when compared tobefore the initiation of the treatment.